US00797 2785B2

(12) United States Patent (10) Patent No.: US 7,972,785 B2 Hsieh et al. (45) Date of Patent: Jul. 5, 2011

(54) BIOMARKERS FOR LIVER FIBROTIC Yi-Chao, Hsu, et al., “Increases in fibrosis-related gene transcripts in INJURY livers of dimethylnitrosamine-intoxicated rats.” J. Biomed Sci. 11:408-417 (2004). Shackel, Nicholas A. et al., “Gene array analysis and the liver.” (75) Inventors: Hui-Chu Hsieh, Changhua (TW); Hepatology, 36(6):1313-1325 (2002). Tzu-Ling Tseng, Chiayi (TW); Li-Jen Utsunomiya, Tohru, et al., “A gene-expression signature can quantify Su, Chiayi (TW); Chi-Ying Huang, the degree of hepatic fibrosis in the rat.” Journal of Hepatology, 41:399-406 (2004). Taipei (TW); Shih-Lan Hsu, Taipei Imaoka, Susumu, et al., “Localization of rat cytochrome P450 in (TW) various tissues and comparison of arachidonic acid metabolism by rat P450 with that by human P450 orthologs.” Drug Metab. (73) Assignees: Industrial Technology Research Pharmacokinet, 2006)478-484 (2005). Institute (ITRI), Hsinchu (TW); Wong, V. S., et al., "Serum hyaluronic acid is a useful marker of liver National Health Research Institutes fibrosis in chronic hepatitis C virus infection.” Journal of viral Hepa titis, 5:187-192 (1998). (NHRI), Miaoli County (TW) Ala-Kokko, L., et al., “Gene expression of type I, III and IV collagens in hepatic fibrosis induced by dimethylnitrosamine in the rat.” (*) Notice: Subject to any disclaimer, the term of this Biochem. J. 244:75-79 (1987). patent is extended or adjusted under 35 Arthur, M.J.P. et al., “Tissue inhibitors of metalloproteinases, hepatic stellate cells and liver fibrosis,” J. Gastroenterol. Hepatol. 13 U.S.C. 154(b) by 326 days. (Suppl.):S33-38 (1998). Bataller, R. and Brenner, D.A., “Liver fibrosis.' J. Clin. Invest. (21) Appl. No.: 11/656,389 115(2):209-218 (2005). Bauer, M. and Schuppan, D., “TGFB1 in liver fibrosis: time to change (22) Filed: Jan. 23, 2007 paradigms?.” FEBS Letts. 502:1-3 (2001). Benyon, R.C. and Arthur, M.J., “Extracellular matrix degradation (65) Prior Publication Data and the role of hepatic stellate cells.” Semin. Liver Dis. 21(3):373-84 (2001). US 2007/O184476A1 Aug. 9, 2007 Day, C.P. “Non-alcoholic steatohepatitis (NASH): where are we now and where are we going?” Gut 50:585-588 (2002). DeSouza, L., et al., “Search for cancer markers from endometrial Related U.S. Application Data tissues using differentially labeled tags iTRAQ and cICAT with mul (60) Provisional application No. 60/761,959, filed on Jan. tidimensional liquid chromatography and tandem mass spectrom etry,” J. Proteome Res.4(2):377-386 (2005). 24, 2006. Dooley, S., et al., “Transforming growth factor f3 signal transduction in hepatic stellate cells via Smad2/3 phosphorylation, a pathway that (51) Int. Cl. is abrogated during in vitro progression to myofibroblasts. TGFB CI2O I/68 (2006.01) signal transduction during transdifferentiation of hepatic stellate GOIN33/53 (2006.01) cells.” FEBS Letts. 502:4-10 (2001). Friedman, S.L., “Liver fibrosis—from bench to bedside.” J. Hepatol. (52) U.S. Cl...... 435/6: 435/7.92 38 (Suppl. 1):S38-53 (2003). (58) Field of Classification Search ...... None Friedman, S.L., “Molecular regulation of hepatic fibrosis, an inte See application file for complete search history. grated cellular response to tissue injury.” J. Biol. Chem. 275(4):2247 2250 (2000). (56) References Cited Friedman, S.L., “Seminars in medicine of the Beth Israel Hospital, Boston: The cellular basis of hepatic fibrosis—Mechanisms and treatment strategies.” N. Engl. J. Med. 328(25): 1828-1835 (1993). U.S. PATENT DOCUMENTS George, J., et al., “Dimethylnitrosamine-induced liver injury in rats: 6,156,501 A 12/2000 McGallet al. the early deposition of collagen.” Toxicol. 156: 129-138 (2001). 2006/0246489 A1* 11/2006 Svetlov et al...... 435/6 Gressner, A.M., et al., “Roles of TGF-B in hepatic fibrosis.” Front. Biosci. 7:d793-807 (2002). FOREIGN PATENT DOCUMENTS EP 1 150 123 A1 10, 2001 (Continued) OTHER PUBLICATIONS Primary Examiner — Sharon Wen (74) Attorney, Agent, or Firm — Finnegan, Henderson, The Merck Manuals Online Medical Library, online). Whitehouse Farabow, Garrett & Dunner LLP Station, NJ: Merck Research Laboratories, 2007. retrieved on Apr. 7, 2009). Retrieved from the Internet: < URL: http://www.merck.com/ (57) ABSTRACT mmpe/print/sec()3/ch026/ch026b.html>. Fibrosis, pp. 1-3.* The present invention provides a method for detecting liver Afdhal Clinical Chemistry 2004, 50: 1299-1300.* fibrotic injury, including fibrosis and/or cirrhosis, by assaying Bast, Jr. et al. Clinical Cancer Research, 2005, vol. 11, pp. 6103 6108. biological samples for differential expression of at least one LaBaer et al. Journal of Proteome Research, 2005, vol. 4, pp. 1053 gene encoding a protein chosen from SEQID NO: 1-SEQID 1059. NO: 63 and human orthologs thereof, wherein differential Baker Nature Biotechnology, 2005, vol. 23, pp. 297-304.* expression of at least one gene Suggests the presence of liver Honda et al. Journal of Autoimmunity 2005, 25:133-140.* fibrosis or cirrhosis. The invention also provides a kit con Ala-Kokko, Leena, et al., “Gene expression of type I, III and IV taining nucleic acid probes or antibodies for detecting liver collagens in hepatic fibrosis induced by dimethylnitrosamine in the fibrosis and/or cirrhosis by assaying the differential expres rat.” Biochem. J., 244:75-79 (1987). sion of proteins encoded by SEQID NO: 1-SEQ ID NO: 63 Rosenberg, William M. C., et al., "Serum markers detect the presence and human orthologs thereof. of liver fibrosis: A cohort study.” Gastroenterology, 127:1704-1713 (2004). 3 Claims, 8 Drawing Sheets US 7,972,785 B2 Page 2

OTHER PUBLICATIONS Mirsalis, J.C., and Butterworth, B.E., "Detection of unscheduled Haggerty, H.G. and Holsapple, M.P., “Role of metabolism in DNA synthesis in hepatocytes isolated from rats treated with dimethylnitrosamine-induced immunosuppression: a review.” genotoxic agents: an in vivo invitro assay for potential carcinogens and mutagens.” Carcinogenesis 1:621-624 (1980). Toxicol. 63:1-23 (1990). Morgan, C.L., Newman, D.J., Burrin, J.M., and Price, C.P. "The Hayaska, A. and Saisho, H., "Serum markers as tools to monitor liver matrix effects on kinetic rate constants of antibody-antigen interac fibrosis.” Digestion 59:381-384 (1998). tions reflect solvent viscosity.” J. Immunol. Meth. 217:51-60 (1998). Hernández-Munoz, R., et al., “Adenosine reverses a preestablished Neubauer, K., et al., "Accumulation and cellular localization of CC1-induced micronodular cirrhosis through enhancing col fibrinogen/fibrin during short-term and long-term rat liver injury.” lagenolytic activity and stimulating hepatocyte cell proliferation in Gastroenterol. 108:1124-1135 (1995). rats.” Hepatol. 34:677-687 (2001). Nielsen, P.E., et al., “Sequence-selective recognition of DNA by Hippo. Y., et al., “Global gene expression analysis of gastric cancer by Strand displacement with a thymine-substituted polyamide.” Science. 254(5037): 1497-1500 (1991). oligonucleotide microarrays.” Cancer Res.62:233-240 (2002). Okita, K., et al., “Current strategies for chemoprevention of Iizuka, N., et al., “Differential gene expression in distinct virologic hepatocellular carcinoma.” Oncology 62 (Suppl. 1):24-28 (2002). types of hepatocellular carcinoma: association with liver cirrhosis.” Sambrook et al., Molecular Cloning. A Laboratory Manual, vol. 3, Oncogene 22:3007-3014 (2003). pp. A8.40-A8.45 and A8.52-A8.55 (3d ed., Cold Spring Harbor Iredale, J.P., “Cirrhosis: new research provides a basis for rational Laboratory Press, Cold Spring Harbor, New York 2001). and targeted treatments.” BMJ 327:143-147 (2003). Schuppan, D., et al., “Hepatitis C and liver fibrosis.” Cell Death Iredale, J.P. "Tissue inhibitors of metalloproteinases in liver fibro Differ. 10:S59-67 (2003). sis.” Int. J. Biochem. Cell Biol. 29(1):43-54 (1997). Seth, D., et al., “Gene expression profiling of alcoholic liver disease Ishak, K., et al., “Histological grading and staging of chronic hepa in the baboon (Papio hamadryas) and human liver.” Am. J. Pathol. titis.” J. Hepatol. 22:696-699 (1995). 163(6):2303-2317 (2003). Jézequel, A.M., et al., "A morphological study of the early stages of Strauss, W.M., "Hybridization With Radioactive Probes.” in Current hepatic fibrosis induced by low doses of dimethylnitrosamine in the Protocols in Molecular Biology 6.3.1-6.3.6. (John Wiley & Sons, rat.” J. Hepatol. 5:174-181 (1987). N.Y. 2000). Ji, J., et al., “Comprehensive analysis of the gene expression profiles Waring, J.F., et al., “Microarray analysis of hepatotoxins in vitro in human gastric cancer cell lines.” Oncogene 21:6549-6556 (2002). reveals a correlation between gene expression profiles and mecha Knodell, R.G., et al., “Formulation and application of a numerical nisms of toxicity.” Toxicol. Letts. 120:359-368 (2001). scoring system for assessing histological activity in asymptomatic Waring, J.F., et al., "Clustering of hepatotoxins based on mechanism chronic active hepatitis.” Hepatol. 1(5):431-435 (1981). of toxicity using gene expression profiles. Toxicol. Appl. Pharmacol. López-De León, A. and Rojkind, M., “A simple micromethod for 175:28-42 (2001). collagen and total protein determination in formalin-fixed paraffin Wright, M.E., et al., “Mass spectrometry-based expression profiling embedded sections.”.J. Histochem. Cytochem. 33(8):737-743 (1985). of clinical prostate cancer.” Mol. Cell. Proteomics 44:545-554 Marvanová, M., et al., “Microarray analysis of nonhuman primates: (2005). validation of experimental models in neurological disorders.” FASEB Zhuang, G., et al., “Measurement of Association Rate Constant of J. 17:929-931 (2003). Antibody-Antigen Interactions in Solution Based on Enzyme-Linked MaZZocca, A., et al., “Expression of transmembrane 4 Superfamily Immunosorbent Assay.” J. Biosci. Bioeng. 92(4):330-336 (2001). (TM4SF) proteins and their role in hepatic stellate cell motility and wound healing migration.” J. Hepatol. 37:322-330 (2002). * cited by examiner U.S. Patent Jul. 5, 2011 Sheet 1 of 8 US 7,972,785 B2

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FIGURE 1B U.S. Patent Jul. 5, 2011 Sheet 3 of 8 US 7,972,785 B2

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Fatty change

FIGURE 2A U.S. Patent Jul. 5, 2011 Sheet 4 of 8 US 7,972,785 B2

B

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A(1-3) 5 (62.5) 3 (27) 4 (44) 0 (0) A(4-6) 3 (36.5) 7 (64) 1 (12) 0 (0) Fibrosis F(0-1) 6 (75) 1 (9) 2 (22) 100 (24) F(2-3). 2 (25) 10 (91) 7 (78) 0 (0) Fatty change - 7 (87.5) 11 (100) 9 (100) 100 (24) + 1 (12.5) 0 (0) 0 (0) 0 (0) n: number of rats FIGURE 2B U.S. Patent Jul. 5, 2011 Sheet 5 of 8 US 7,972,785 B2

Control Treatment

FIGURE 3A U.S. Patent Jul. 5, 2011 Sheet 6 of 8 US 7,972,785 B2

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FIGURE 4 U.S. Patent Jul. 5, 2011 Sheet 8 of 8 US 7,972,785 B2

FIGURES US 7,972,785 B2 1. 2 BOMARKERS FOR LIVER FIBROTC The invention provides a method of detecting liver fibrosis INJURY and/or cirrhosis comprising obtaining a biological sample from apatient and assaying the sample for differential expres CROSS REFERENCE TO RELATED sion of at least one gene encoding a protein chosen from SEQ APPLICATIONS ID NO: 1-SEQ ID NO: 63 and human orthologs thereof, wherein the differential expression of at least one gene Sug The present invention is related to and claims the benefit of, gests the presence of liver fibrosis and/or cirrhosis. under U.S.C. S 119(e), U.S. provisional patent application Also provided is a method of detecting liver fibrosis and/or Ser. No. 60/761,959, filed on Jan. 24, 2006, which is cirrhosis by assaying the biological sample for differential expressly incorporated fully herein by reference. 10 expression of the human orthologs described above, wherein the human orthologs comprise at least one gene encoding a FIELD OF THE INVENTION protein chosen from SEQID NO: 64-SEQID NO: 120. Also provided is a kit for diagnosing liver fibrosis and/or Herein described are methods of detecting liver fibrosis cirrhosis, which comprises one or more nucleic acid probes and/or cirrhosis based on the differential expression of vari 15 that hybridize to nucleic acid molecules of at least one gene ous proteins, and kits for diagnosing fibrosis and/or cirrhosis. encoding a protein chosen from SEQID NO: 1-SEQID NO: 63 and human orthologs thereof and packaging indicating use BACKGROUND for detection of liver fibrosis and/or cirrhosis. In addition, a kit is provided for diagnosing liver fibrosis Liver fibrosis represents a continuous disease spectrum comprising one or more nucleic acid probes as described characterized by an increase in total liver collagen and other above, wherein the human orthologs comprise at least one matrix proteins that disrupt the architecture of the liver and gene encoding a protein chosen from SEQIDNO: 64-SEQID impair liver function (1,2). The progression offibrosis in the NO: 120. liver is a response to necroinflammatory changes. The overall The invention further provides a kit for diagnosing liver liver fibrosis process is one of dynamic inflammation and 25 fibrosis and/or cirrhosis that comprises one or more antibod repair and has the potential to be resolved (3). Fibrosis is seen ies that specifically bind to at least one protein encoded by a as scar formation in the patient’s liver. When the liver sequence chosen from SEQ ID NO: 1-SEQ ID NO: 63 and becomes permanently injured and scarred, the condition is human orthologs thereof, and packaging indicating use for called cirrhosis. detection of liver fibrosis and/or cirrhosis. Liver fibrosis and/or cirrhosis are the major risk factors of 30 Also provided is a kit for diagnosis of liver fibrosis and/or hepatocellular carcinoma (HCC). A range of factors, such as cirrhosis comprising one or more antibodies as described hepatitis B virus (HBV), hepatitis C virus (HCV), hepatotox above, wherein the human orthologs comprise at least one ins, metabolic disorders and alcoholism, can induce both liver gene encoding a protein chosen from SEQIDNO: 64-SEQID fibrosis and/or cirrhosis, which share similar phenotypes NO: 120. (3–7), with cirrhosis being the end-stage of fibrosis. However, 35 The invention also provides a method of identifying a it is not clear what types of genes are involved or how they act compound that decreases the differential expression of at when liver injury and repair occur. Moreover, the cirrhosis least one gene-encoding a protein chosen from SEQID NO: caused by these risk factors often progresses insidiously. 7-SEQID NO: 23 and SEQID NO: 42-SEQID NO: 63 and Patients with end-stage liver cirrhosis can die within one year human orthologs thereof, comprising providing a cell unless they accept liver transplantation, which has a 75% 40 expressing at least one of the aforementioned genes, contact five-year survival rate (3). ing the cell with a test compound, and determining whether Previous biochemical studies have reported that there are the differential expression of the at least one gene is decreased 39 well-known fibrosis or cirrhosis markers (3,8,9), but some in the presence of the test compound, wherein the decreased markers are obtained through invasive sampling. differential expression is an indication of halting or reversing Studies for additional markers have proceeded based on 45 liver fibrosis and/or cirrhosis. microarray analysis of transcriptomes as well as quantitative The invention further provides a method of identifying a proteomics. Microarray technologies have been widely used compound that increases the differential expression of at least for comprehensive gene expression analysis. In particular, one gene encoding a protein chosen from SEQID NO: 1-SEQ large-scale microarray analysis of gene expression enables ID NO: 6 and SEQID NO: 24-SEQ ID NO: 41 and human researchers to analyze simultaneous changes in thousands of 50 orthologs thereof, comprising providing a cell expressing at genes and identify significant patterns (10, 11). To date, the least one of the aforementioned genes, contacting the cell most widely used technologies in differential proteomics with a test compound, and determining whether the differen research were two-dimensional gel electrophoresis (2DE) tial expression of the at least one gene is increased in the and liquid chromatography-based isotope-coded affinity tag presence of the test compound, wherein the increased differ ging (ICAT) technologies (12). Recently, a variation of the 55 ential expression is an indication of halting or reversing liver ICAT technology, iTRAQ (isobaric tags for relative and abso fibrosis and/or cirrhosis. lute quantitation), has been introduced. Both ICAT and Additional objects and advantages of the invention will be iTRAOtagging permit online identification of multiple mark set forth in part in the description which follows, and in part ers and relative quantification of these proteins. will be obvious from the description, or may be learned by 60 practice of the invention. The objects and advantages of the SUMMARY OF THE INVENTION invention will be realized and attained by means of the ele ments and combinations particularly pointed out in the The inventors have used differential analysis such as appended claims. microarray mRNA expression profiling and quantitative pro It is to be understood that both the foregoing general tein profiling to find additional unique and identifiable signa 65 description and the following detailed description are exem tures potentially valuable for the diagnosis and treatment of plary and explanatory only and do not limit the invention as liver fibrosis and/or cirrhosis. claimed. US 7,972,785 B2 3 4 The accompanying drawings, which are incorporated in log scale on the right side of the plot. The expression levels of and constitute a part of this specification, illustrate several tWO Timp1 transcripts, rc AI169327 at and embodiments of the invention and together with the descrip rc AI169327 g at (marked by circle and triangle), are rela tion, serve to explain the principles of the invention. tive to the mean of all gene expression levels, and are mea sured by the scale indicated on the left side of the plot. SUMMARY OF THE TABLES FIG. 4A shows the vimentin expression ratios in DMN treated and control rats at 2, 4, and 6 weeks as determined by Table 1 is the result of a biochemical analysis of the serum iTRAQ proteomics. Vimentin expression increased 2.4- to ofrats treated with dimethylnitrosamine (DMN) as compared 3.4-fold in the liver when rats were treated with DMN. with that of the control rats over a six-week time period. 10 FIG.4B shows the result of Western blot analysis of DMN Table 2 is a list of genes showing significant changes in induced vimentin expression. The observed result matched gene expression and their human orthologs. The genes were with the trend measured by mass spectrometry in iTRAQ selected from the combined results of microarray analysis proteomic experiments. and quantitative proteomic analysis and those whose relation FIG. 5 shows the result of Western blot analysis comparing ship to fibrosis are not yet disclosed in public literature. 15 the expression of carbonic anhydrase I in human Sera from Table 3 is the result of interacting network analysis for the normal volunteers and from patients with hepatitis B-related proteins showing significant changes in expression in oligo cirrhosis. nucleotide microarray (Table 3A) and iTRAQ proteomic (Table 3B) studies. Proteins in the network identified in the DESCRIPTION OF THE EMBODIMENTS present study which have not previously been reported to associate with liver injury are shown in bold. Definitions BRIEF DESCRIPTION OF THE DRAWINGS As used herein, the term “biological sample” refers to any biological material collected from cells, tissues, or organs of FIG. 1A is a schematic illustration of DMN treatment for 25 the Subject. The source of the biological sample may vary inducing fibrosis in rats. Each rat was either injected with depending on the particular symptoms present in the Subject DMN three times per week for three consecutive weeks to be diagnosed. The biological sample may be analyzed (shown as inverted triangles) or with normal saline as control. immediately after it is taken, or stored. If stored, the sample Rats were weighed and sacrificed each week (shown as tri may be equilibrated with an appropriate storage buffer, and angles, starting on day 11, which is referred to as first week, 30 kept at 4°C., at -20°C., at -70° C., or even in cryogenic until week six). Blood samples were collected for biochemi liquids, Such as liquid nitrogen or liquid helium. In one cal assays (summary in Table 1) and livers were excised and embodiment, the biological sample may consist of blood, weighed. Livers were then either fixed in formaldehyde for serum, or plasma. In another embodiment, the biological histopathology or used to isolate RNA and protein for sample may consist of a biopsy or tissue sample. In additional microarray and iTRAO proteomic studies, respectively. 35 embodiments of the invention, the biological sample may FIG. 1B is the quantitative real-time PCR (Q-RT-PCR) consist of aminotic fluid, milk, Saliva, cerebrospinal fluid, result of TgfB1 (transforming growth factor-f1) in DMN lymph, Sweat, mucus, synovial fluid, lacrimal fluid, or other treated rats, showing a higher level of TgfB1 expression, the clinical specimens or samples. strongest known inducer of fibrogenesis. As used herein, the term “patient” refers to a mammalian FIG. 2A shows the histopathological examination results 40 animal, including but not limited to human, primates, domes of liver tissues from DMN-treated rats. The representative tic mammals, laboratory mammals, etc. phenotype of the rat liver tissue was characterized by scoring As used herein, the term “differential expression” refers to the four histopathological features as follows: the necrosis gene expression on the RNA/mRNA level, protein level, or scores are from NO to N3 (the first row), the inflammation both RNA/mRNA and protein levels as compared to normal scores are from IO to I3 (the second row), the fibrosis scores 45 gene expression, e.g., an increased or decreased gene expres are from F0 to F3 (the third row), and the fatty change scores sion on the RNA/mRNA level, protein level, or both RNA/ are presence or absence (+ and -) in the last row. mRNA and protein levels. FIG. 2B is a summary of the histopathological scores for As used herein, the terms 'gene' and "gene encoding a the rat model. The results are ranked by time course. The protein’ refer to any nucleic acid sequence or portion thereof necroinflammatory change is divided into three grades: 50 with a functional role in encoding or transcribing a protein. A0-none, A(1-3) mild and A(4-6)-moderate necroinflam The gene may encompass all the nucleic acids responsible for matory. The fibrosis change is divided into two grades: F(0- encoding a functional protein of certain SEQID NO or only 1)-normal to fibrous expansion of portal tracts, F(2-3)= a portion of the nucleic acids responsible for encoding or bridge fibrosis to frequent bridging fibrosis with nodule for expressing a protein of certain SEQID NO. The nucleic acid mation. The fatty change is shown as presence or 55 sequence may contain normal sequences as well as genetic absence (+/-). The number of rats is counted and used to abnormalities within exons, introns, initiation or termination calculate the percentage of rats in each histopathological level regions, promoter sequences, other regulatory sequences or at each time point. unique adjacent regions to the gene. FIG. 3A shows the dendrogram of two hundred and fifty As used herein, the term “human ortholog” refers to a six (256) gene expression patterns from the DMN-induced 60 corresponding human gene wherein the non-human gene and fibrosis model in rats. the human gene are derived from a single ancestral gene in the FIG. 3B shows the comparison of Timp1 (tissue inhibitor last common ancestor of human and non-human, and the of metalloproteinase 1) expression between the Q-RT-PCR human genes have the same function. result and the microarray data, and a good concordance As used herein, the term “probes’ refers to hybridization between the two results is shown. For the Q-RT-PCR assay of 65 probes that are oligonucleotides that bind in a base-specific Timp 1, expression levels (marked by the square) are relative manner to a complementary strand of nucleic acid. Such to the mean of all gene expression, and are measured by the probes also include peptide nucleic acids, as described in US 7,972,785 B2 5 6 Nielsen et al., 1991, (13), and other nucleic acid analogs and with cyclic, bicyclic, depsicyclic, or depsibicyclic peptide nucleic acid mimetics. See, e.g., U.S. Pat. No. 6,156,501, backbones, single chain protein as well as multimers, as well which is incorporated herein by reference. as any fragment or portion of the intact protein molecule. As used herein, the term “nucleic acid refers to any DNA As used herein, the term “compound” refers to a substance or RNA/mRNA, for example, chromosomal, mitochondrial, comprising one or more chemical elements in any proportion viral and/or bacterial nucleic acid present in tissue sample as and of any structure, including but not limited to cyclic, well as synthetic nucleic acids. The term “nucleic acid bicyclic, branched or straight chain. The compound may be encompasses either or both strands of a double stranded organic or inorganic. The compound also refers to a compo nucleic acid molecule and includes any fragment orportion of sition comprising one or more chemical elements, one or an intact nucleic acid molecule. 10 more herbal/plant elements or herbal/plant extractions, or As used herein, the term "packaging refers generally to both chemical elements and herbal/plant elements or extrac packaging material comprising external labeling and internal tions. material in the container, including but not limited to instruc tions for using the kit. EMBODIMENTS As used herein, the term “antibody.” refers to an immuno 15 globulin or a fragment or a derivative thereof, and encom Chronic liver disease is a common and potentially lethal passes any polypeptide comprising an antigen-binding site, problem in Asia. The development of hepatocellular carci whether produced in vitro or in vivo. The term includes, but is noma (HCC) is generally preceded by hepatic cirrhosis, not limited to, polyclonal, monoclonal, monospecific, which occurs at the end stage of fibrosis. The same proteins polyspecific, humanized, single-chain, chimeric, synthetic, are expressed in both cirrhosis and fibrosis. Changes in gene recombinant, hybrid, mutated, and grafted antibodies. The expression of these proteins during liver fibrosis are exam term “antibody also includes antibody fragments such as ined to identify markers of liver fibrosis to assist in the diag Fab, F(ab'). Fv, scEv, Fd, dAb, and other antibody fragments nosis of fibrosis and/or cirrhosis. The study begins with the that retain antigen-binding function, i.e., the ability to bind a establishment of a liver fibrosis model. Dimethylnitrosamine specific antigen. Typically, such fragments would comprise 25 (DMN), a non-genotoxic hepatotoxin, is used to induce rat an antigen-binding domain, i.e., a part of an antibody mol necroinflammatory and hepatic fibrosis, as described in Jeze ecule that comprises amino acids responsible for the specific quel A. M. et al. (14), which is a known model for studying binding between the antibody and the antigen. An antigen human liver damage (15). During a six-week time course, binding domain typically comprises an antibody light chain histopathological, biochemical and quantitative RT-PCR variable region (V) and an antibody heavy chain variable 30 analyses confirmed the incidence of hepatic fibrosis in the rat region (V), however, it does not necessarily have to com model system. prise both. For example, a so-called Fd antibody fragment The microarray and the iTRAQ quantitative proteomics consists only of a V. domain, but still retains some antigen technology were used. The iTRAO technology permits the binding function of the intact antibody. identification of multiple proteins at the same time as well as As used herein, the term “specifically binds or the like, 35 the relative quantification of these proteins. Applied Biosys means that two molecules form a complex that is relatively tems iTRAQ Reagents are a multiplexed set of four isobaric stable under physiologic conditions (e.g., a stable antigen/ reagents. The four reagents are amine-specific and yield antibody complex). The term is also applicable where, for labeled peptides. The labeled peptides are identical in mass example, an antigen-binding domain is specific for a particu and hence also identical in single MS mode. They produce lar epitope, which is found on a number of molecules. Thus, 40 strong, diagnostic, low-mass MS/MS signature ions allowing an antibody may specifically bind multiple proteins when it for quantification of up to four different samples simulta binds to an epitope present in each. Specific binding is char neously. Quantification is performed via the differences in acterized by a selective interaction, often including high abundance of four productions, i.e., productions weighing affinity binding with a low to moderate capacity. Nonspecific 114, 115, 116, and 117 daltons that are each cleaved from one binding is usually a less selective interaction, and may have a 45 of the four possible tags. Since all peptides are tagged, pro low affinity with a moderate to high capacity. Typically, bind teome coverage is expanded and analysis of multiple peptides ing is considered specific when the affinity is at least 10 M', per protein improves the confidence in those identified (16). 10M, 107M" or 10 M'. If necessary, non-specific bind The multi-sample capability of the iTRAQ technology pro ing can be reduced without Substantially affecting specific vides a way to compare the protein expression profile of binding by varying the binding conditions. Such conditions 50 different liver states simultaneously. are known in the art, and a skilled artisan using routine tech The model employed in the present study is very similar to niques can select appropriate conditions. The conditions are the human liver study model, and thus findings from this usually defined interms of concentrations of antibodies, ionic study can have human clinical applications. This study strength of the solution, temperature, time allowed for bind employed DMN, a potent non-genotoxic hepatotoxin, to ing, concentration of non-related molecules (e.g., blocking 55 simulate liver fibrosis (17, 18). DMN has been demonstrated agents such as serum albumin or milk casein), and so forth. to induce liver damage rapidly and also has been empirically See, e.g., Morgan et al., “The Matrix Effects on Kinetic Rate proven to be useful for the study of human fibrosis formation Constants of Antibody-Antigen Interactions Reflect Solvent (14-15, 19) as mentioned above. Also, the serum markers that Viscosity.” J. Immunol. Meth. 217:51:60 (1998); and Zhuang showed significant differences in expression in the rat liver et al., “Measurement of Association Rate Constant of Anti 60 fibrosis model established in this study as compared to the body-Antigen Interactions in Solution Based on Enzyme controls, see Table 1, are the same serum markers for human LinkedImmunosorbent Assay.J. Biosci. Bioeng. 92(4): 330 liver fibrosis, e.g., the thirteen serum markers of albumin, 336 (2001). glutamic oxaloacetic transaminase (GOT), glutamic pyruvic As used herein, the term “protein’ refers to a polymeric transaminase (GPT), total bilirubin, alkaline phosphatase form of any length of amino acids, which can include natu 65 (AKP), acid phosphatases (ACP), C.-fetoprotein (AFP), blood rally-occurring or synthetic amino acids and coded and non urea nitrogen (BUN), cholesterol (CHOL), lactate dehydro coded amino acids, peptides, depsipeptides, polypeptides genase (LDH), globulin, prothrombin time (PT) and blood US 7,972,785 B2 7 8 platelets (PLT). Thus, the markers identified from this study yet another embodiment, the invention relates to assaying for can be used to diagnose liver fibrosis and/or cirrhosis in the decreased expression of at least one gene encoding a humans. protein chosen from the humanorthologs comprising SEQID Detection Methods NO:64-SEQIDNO: 67 and SEQIDNO: 83-SEQID NO: 99. The present study identified nearly 97 genes as biomarkers The invention also provides a clustering of the genes by for liver fibrotic injury. Results of a PubMed literature search functional groups. Thus, the invention provides a method of indicate that approximately 30% of the differentially detecting liver fibrosis and/or cirrhosis comprising assaying a expressed genes identified in this study by microarray and biological sample for differential expression of at least one proteomics approach had been proven to be related to fibrosis gene encoding a protein that functions in cancer, cell cycle, or cirrhosis. The remaining 70% are not yet reported in any 10 and cell morphology chosen from sequences comprising SEQ literature. Combining all the information obtained by both ID NO:9, SEQID NO: 69, SEQID NO: 12, SEQID NO: 72, microarray and quantitative proteomics technology, sixty SEQ ID NO: 4, SEQ ID NO: 65, SEQ ID NO: 17, SEQID three (63) genes were selected, all of which showed signifi NO: 76, SEQ ID NO: 7, SEQID NO: 68, SEQ ID NO: 19, cant changes in expression between DMN-treated and DMN SEQ ID NO: 78, SEQID NO. 5, SEQ ID NO: 66, SEQID untreated groups, but have not been previously reported in 15 NO: 20, SEQID NO: 79, SEQID NO: 22, SEQID NO: 81, any literature in relation to liver fibrosis or cirrhosis (Table 2). SEQID NO: 21, and SEQID NO: 80; a protein that functions The 63 genes in DMN-treated rats have a difference in gene in lipid metabolism, Small molecule biochemistry, organis expression on the RNA/mRNA level and/or the protein level mal injury and abnormalities chosen from sequences com when compared to normal gene expression. Some genes are prising SEQID NO: 10, SEQID NO: 70, SEQID NO:3, SEQ up-regulated while others are down-regulated. Those 63 rat ID NO: 64, SEQID NO:14, SEQID NO: 73, SEQID NO: 15 genes were converted into their human ortholog genes and SEQID NO: 74; a protein that functions in hematological according to the ortholog assertions from the EnsEMBL and disease, endocrine system development and function, ner HomoloGene database (Table 2 SEQ ID NO: 1-SEQ ID Vous system development and function comprising SEQID NO: 120). NO: 13; a protein that functions in cancer, cell morphology, Thus the invention provides a method for detecting liver 25 dermatological diseases and conditions chosen from fibrotic injury based on the liver injury-related differential sequences comprising SEQID NO: 8, SEQID NO: 44, SEQ gene expression study described herein. In the detection ID NO: 102, SEQID NO:45, SEQID NO: 103, SEQID NO: method, differential expression of a gene encoding a protein 48, SEQID NO: 106, SEQID NO:31, SEQID NO:90, SEQ includes differential expression of the full length gene encod ID NO:35, SEQ ID NO: 93, SEQID NO:50, SEQID NO: ing a full length protein or a portion of the gene encoding a 30 108, SEQID NO:51, SEQID NO: 109, SEQID NO:40, and portion of the protein. SEQID NO: 98; a protein that functions in lipid metabolism, Accordingly, the invention provides a method of detecting small molecule biochemistry, and molecular transport chosen liver fibrosis and/or cirrhosis comprising obtaining a biologi from sequences comprising SEQID NO:25, SEQID NO: 84, cal sample from a patient, and assaying the sample for differ SEQID NO:42, SEQID NO: 100, SEQID NO: 27, SEQ ID ential expression of at least one gene encoding a protein 35 NO: 86, SEQID NO:47, SEQID NO: 105, SEQID NO:36, chosen from SEQ ID NO: 1 to SEQ ID NO: 63 and human SEQID NO: 94, SEQ ID NO: 16, SEQID NO: 75, SEQ ID orthologs thereof, wherein the differential expression of at NO:53, SEQID NO: 11, SEQID NO:37, SEQID NO: 95, least one gene Suggests the presence of liver fibrosis and/or SEQID NO:38, SEQID NO:96, and SEQID NO: 107; and cirrhosis. In another embodiment, the invention provides a a protein that functions in cancer, cellular movement, cellular method of detecting liver fibrosis and/or cirrhosis by assaying 40 growth and proliferation chosen from sequences comprising the sample for differential expression of at least one gene SEQID NO:59, SEQID NO: 117, SEQID NO:46, SEQ ID encoding a protein chosen from the human orthologs com NO: 104, SEQID NO:30, SEQID NO: 89, SEQID NO:52, prising SEQID NO: 64 to SEQID NO: 120. SEQID NO: 110, SEQID NO:58, SEQID NO: 116, SEQID Human orthologs of proteins identified in this study can be NO:55, SEQID NO: 113, SEQID NO:39, SEQID NO:97, derived from the search using gene/protein on the Ensembl 45 SEQID NO:56, SEQID NO: 114, SEQID NO:57, and SEQ database together with the HomoloGene NCBI database. ID NO: 115. In the practice of this invention, differential gene expres Diagnostic Kits sion may be assayed by transcription analysis or quantitative The invention also provides diagnostic kits based on the proteomic analysis such as microarray, Q-RT-PCR, ICAT and genes and/or proteins described above. In one embodiment, iTRAO, or any other appropriate methods known to one 50 there is a kit for diagnosing liver fibrosis and/or cirrhosis that skilled in the art. comprises one or more nucleic acid probes that hybridize to In another embodiment, the invention relates to assaying nucleic acid molecules of at least one gene encoding a protein the up-regulation or increased expression of one or more marker chosen from SEQ ID NO: 1-SEQ ID NO: 63 and genes encoding a protein chosen from SEQID NO: 7-SEQID human orthologs thereof and packaging indicating the use for NO: 23 and SEQ ID NO: 42-SEQ ID NO: 63 and human 55 detecting liver fibrosis. The kit can detect the differential orthologs thereof, wherein the increased expression Suggests expression, e.g., the up-regulation or down-regulation, of the presence of liver fibrosis and/or cirrhosis. In yet another these genes, wherein the differential expression would Sug embodiment, the invention relates to assaying for the gest liver fibrosis and/or cirrhosis. In another embodiment, increased expression of at least one gene encoding a protein the invention also provides a kit for diagnosing fibrosis and/or chosen from the human orthologs comprising SEQ ID NO: 60 cirrhosis comprising nucleic acid probes that hybridize to 68-SEQID NO: 82 and SEQID NO: 100-SEQID NO: 120. nucleic acid molecules of at least one gene encoding a protein The invention also provides, in another embodiment, marker chosen from SEQ ID NO: 1-SEQ ID NO: 63 and assaying for the down-regulation or decreased expression of human orthologs thereof. The differential expression of the one or more genes encoding a protein chosen from SEQID genes would suggest fibrosis and/or cirrhosis. In another NO: 1-SEQID NO: 6 and SEQID NO: 24-SEQID NO: 41 65 embodiment, the diagnostic kit comprises one or more and human orthologs thereof, wherein the decreased expres nucleic acid probes that hybridize to nucleic acid molecules sion suggests the presence of liver fibrosis and/or cirrhosis. In of at least one gene encoding the human orthologs comprising US 7,972,785 B2 9 10 proteins chosen from SEQID NO: 64-SEQ ID NO: 120 for NO: 63 and human orthologs thereof, and further comprises diagnosing liver fibrosis and/or cirrhosis. packaging that indicates use for detection of liver fibrosis The kit may be prepared by techniques known to one and/or cirrhosis. In an alternative embodiment, the kit com skilled in the art. By way of example, the probes may be prises one or more antibodies that specifically bind to at least labeled, using labeling techniques that are known to one one human ortholog protein encoded by a sequence chosen skilled in the art, to facilitate detection, including but not from SEQ ID NO: 64-SEQ ID NO: 120, and further com limited to radioisotope labels or fluorescent labels. The prises packaging that indicates use for detection of liver fibro probes can hybridize to nucleic acid molecules that are either sis and/or cirrhosis. Differential expression of these genes or both strands of a double stranded nucleic acid molecule and Suggests the presence of fibrosis and/or cirrhosis. include any fragment or portion of an intact nucleic acid 10 The antibodies specific to the proteins may be obtained by molecule. monoclonal or polyclonal techniques known to one skilled in Nucleic acid hybridization is affected by such conditions as the art. The antibody can be labeled with radioisotopes such salt concentration, temperature, or organic solvents, in addi as S, ''C, ‘I, H, and 'I, using the techniques described tion to the base composition, length of the complementary in Current Protocols in Immunology (20), as an example. The Strands, and the number of nucleotide base mismatches 15 radioactivity can be measured using Scintillation counting. between the hybridizing nucleic acids, as will be readily The antibody may also be fluorescently labeled by, for appreciated by those skilled in the art. Stringency conditions example, rare earth chelates (europium chelates), Texas Red, depend on the length and base composition of the nucleic rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, acid, which can be determined by techniques well known in phycocrytherin, phycocyanin, or commercially available the art. Generally, Stringency can be altered or controlled by, fluorophores such SPECTRUM ORANGER) and SPEC for example, manipulating temperature and salt concentra TRUMGREENR) and/orderivatives of any one or more of the tion during hybridization and washing. For example, a com above. The fluorescent labels can be conjugated to the anti bination of high temperature and low salt concentration body using the techniques disclosed in Current Protocols in increases stringency. Such conditions are known to those Immunology, Supra., for example. Fluorescence can be quan skilled in the art and can be found in, for example, Strauss, W. 25 tified using a fluorimeter. M. “Hybridization With Radioactive Probes,” in Current Pro Differential protein expression may be assayed using anti tocols in Molecular Biology 6.3.1-6.3.6. (John Wiley & Sons, bodies with commonly used methods known in the art, such N.Y. 2000). Both aqueous and nonaqueous conditions as as Western blotting or enzyme-linked immunosorbent assay described in the art can be used. (ELISA). Western blotting begins with an electrophoresis An example of Stringent hybridization conditions is 30 step, where proteins from a biological sample of interest are hybridization in 0.1xSSC (15 mM sodium chloride/1.5 mM separated on the basis of size and electromagnetic charge by sodium citrate) at 50° C. or higher. Another example of strin sodium dodecyl sulfate-polyacrylamide gel electrophoresis gent hybridization conditions is hybridization overnight at (SDS-PAGE), according to standard methods known in the 42°C. in 50% formamide, 1xSSC (150 mM. NaCl, 15 mM art. See, e.g., SAMBROOKETAL, 3 MOLECULAR CLONING: A LABO sodium citrate), 50 mM sodium phosphate (pH 7.6), 5xDen 35 RATORY MANUAL A8.40-A8.45 (2001) (describing various hardt’s solution, 10% (w/v) dextran sulfate, and 20 ug/ml reagents and methods for electrophoresis of proteins by SDS denatured, sheared salmon sperm DNA, followed by washing PAGE). The contents of the gel are then transferred to nitro in 0.1 xSSC at about 65° C. Highly stringent conditions can cellulose, nylon, PVDF, or other membrane or filter suitable include, for example, aqueous hybridization (e.g., free of for fixation and Western blotting by standard methods also form amide) in 6xSSC (where 20xSSC contains 3.0 MNaCl 40 known in the art. The transfer may be by immersion, semi-dry and 0.3 M sodium citrate), 1% (w/v) sodium dodecyl sulfate blotting, or by other comparable methods known in the art. (SDS) at 65° C. for about 8 hours (or more), followed by one Next, the filters or membranes are fixed to prevent loss of the or more washes in 0.2xSSC, 0.1% SDS at 65° C. target proteins during the several hybridization, washing, and Moderately stringent hybridization conditions permit a staining steps comprising Western blotting. Fixation may be nucleic acid to bind a complementary nucleic acid that has at 45 accomplished by heat, cross-linking with ultraviolet light, or least about 60%, at least about 75%, at least about 85%, or by other comparable methods known in the art. See, e.g., greater than about 90% identity to the complementary nucleic SAMBROOKETAL, 3 MOLECULAR CLONING: A LABORATORY MANUAL acid. Stringency of hybridization is generally reduced by A8.52-A8.55 (describing various reagents and methods for decreasing hybridization and washing temperatures, adding immunoblotting and detection of antigen/antibody com formamide to the hybridization buffer, or increasing salt con 50 plexes). centration of the washing buffer, either individually or in Non-specific antibody binding sites on the fixed filter or combination. Moderately stringent conditions can include, membrane are blocked with buffered solutions (e.g., phos for example, aqueous hybridization (e.g., free of formamide) phate-buffered saline (“PBS) or the like) containing a block in 6xSSC, 1% (w/v) SDS at 65° C. for about 8 hours (or ing agent Such as, for example, 0.5% (w/v) low-fat dry milk or more), followed by one or more washes in 2xSSC, 0.1% SDS 55 5% (w/v) bovine serum albumin (BSA). After blocking, the at room temperature. Another exemplary hybridization under filter or membrane then undergoes the primary antibody incu moderate stringency comprises hybridization in 6xSSC, bation. After primary antibody incubation, the filter or mem 5xDenhardt’s reagent, 0.5% (w/v) SDS, and optionally 100 brane is washed, and the presence of antibody-antigen com ug/ml Sonicated salmon or herring sperm DNA, at about 42 plexes detected using a secondary antibody labeled with C., followed by washing in 2xSSC, 0.1% (w/v) SDS at 65° C. 60 chromogenic, fluorogenic, or chemiluminescent means. Anti Other permutations and possibilities will be readily apparent body-antigen complexes are then detected colorimetrically to those of ordinary skill in the art, and are considered as (e.g., with horseradish peroxidase and TMB), or by autorad equivalents within the scope of the instant invention. iography (e.g., alkaline phosphatase). If detected colorimetri In yet another embodiment, the invention provides a kit for cally, or by chemiluminescence, the amount of color of fluo diagnosing liver fibrosis and/or cirrhosis comprising one or 65 rescence may be measured using a luminometer, a more antibodies that specifically bind to at least one protein spectrophotometer, or other similar instruments. If detected encoded by a sequence chosen from SEQID NO: 1-SEQID autoradiographically, the amount of bound antibody may be US 7,972,785 B2 11 12 measured from the exposed X-ray film using a densitometer, antibody labeled with chromogenic (e.g., horseradish peroxi or similar instrument. See, e.g., SAMBROOKETAL, 3 MOLECULAR dase and TMB), fluorescent or chemiluminescent (e.g., alka CLONING: A LABORATORY MANUAL A8.52-A8.55. line phosphatase) means. See, e.g., id. at 15-21 (discussing Secondary antibodies used in Western blotting, whether antibody preparation and use, as well as commonly used polyclonal or monoclonal, may be labeled with a ligand (Such detection molecules). The amount of color or fluorescence as biotin) or a detectable marker (Such as a fluorescent group may be measured using a luminometer, a spectrophotometer, or an enzyme) using conventional techniques. Suitable labels or other similar instruments. There are many common varia include fluorophores, chromophores, electron-dense reagents tions on the standard ELISA protocol, including competitive (e.g., silver or gold), enzymes, and ligands having specific ELISA, sandwich ELISA, and numerous others. One of ordi binding partners. Enzymes such as horseradish peroxidase or 10 nary skill in the art will select the appropriate protocol to use, alkaline phosphatase are typically detected by their activity. depending on the antigen to be detected, the Source of antigen For example, horseradish peroxidase can be detected by its and/or primary antibody used in the assay, and any other ability to convert tetramethylbenzidine (TMB) to a blue pig relevant experimental parameters. These and many other per ment, quantifiable with a spectrophotometer. Other suitable mutations will be readily apparent to those of ordinary skill in ligands and/or detectable markers include bioting and avidin 15 the art, are considered as equivalents within the scope of the or streptavidin, IgG and protein A, and the numerous addi invention. tional receptor-ligand couples known in the art. Other permu Screening Methods tations and possibilities will be readily apparent to those of Also described is a method that identifies a compound that ordinary skill in the art, and are considered as equivalents would decrease the differential expression of at least one gene within the scope of the instant invention. encoding a protein chosen from SEQID NO: 7-SEQID NO: An ELISA begins with an antigen adsorption step, where 23 and SEQID NO:42-SEQID NO: 63 and human orthologs the target antigen or antigens are adsorbed to the wells of a thereof. The method provides a cell expressing at least one of microtiter plate. See, e.g., KIERKEGAARD & PERRY LABORATO these genes, contacting the cell with a test compound to RIES, INC., TECHNICAL GUIDE FOR ELISA 9-13 (2003). The most determine whether the differential expression is decreased in commonly used adsorption buffers for antibodies are 50 mM 25 the presence of the test compound, wherein the differential Carbonate, pH=9.6; 10 mM Tris-HCl, pH=8.5; and 10 mM expression is an indication of halting or reversing liver fibro PBS, pH=7.2. These buffers work well for many proteins. If sis and/or cirrhosis. The same method can also identify a the target antigens are not readily adsorbed to the Surface of compound that would increase the differential expression of the microtiter plate, plates with surfaces modified or deriva at least one gene encoding a protein chosen from SEQID NO: tized to permit covalent linkage of proteins to their surface by 30 1-SEQID NO: 6 and SEQID NO: 24-SEQID NO: 41 and a variety of chemical means are widely available from com human orthologs thereof. mercial suppliers. Time and temperature are the most impor Other embodiments of the invention will be apparent to tant factors affecting the amount of protein adsorbed. those skilled in the art from consideration of the specification Once the wells of a microtiter plate are coated with the and practice of the invention disclosed herein. It is intended desired antigen or antigens, they are washed with a blocking 35 that the specification and examples be considered as exem buffer to block non-specific antibody binding and to mini plary only, with the true scope and spirit of the invention being mize false positive results. See, e.g., id. at 13-14 (discussing indicated by the following claims. methods and reagents for blocking microtiter plates). Com Unless defined otherwise, the meanings of all technical and monly used blocking agents are either protein solutions, such Scientific terms used herein are those commonly understood as BSA (typically used at concentrations between 1% and 5% 40 by one of ordinary skill in the art to which this invention (w/v) in PBS, pH-7.0), non-fat dry milk, casein (the main belongs. One of ordinary skill in the art will also appreciate protein component of non-fat dry milk), or caseinate (a more that any methods and materials similar or equivalent to those soluble version of casein, produced by partial digestion with described herein can also be used to practice or test the inven Sodium hydroxide), normal serum (typically used at concen tion. Further, all publications mentioned herein are incorpo trations between 1% and 5% (v/v)), and gelatin (normally 45 rated by reference. used at concentrations between 1% and 5% (w/v)), or non With respect to ranges of values, the invention encom ionic detergents, such as TWEEN-20TM and TRITON passes the upper and lower limits and each intervening value X-100TM. between the upper and lower limits of the range to at least a Washing reagents are selected for their ability to disrupt tenth of the upper and lower limits unit, unless the context low-affinity interactions between various reaction compo 50 clearly indicates otherwise. Further, the invention encom nents that can affect the ability to detect specific antigen passes any other stated intervening values. antibody interactions. See, e.g., id. at 14-15 (discussing meth Further, all numbers expressing quantities of ingredients, ods and reagents for washing microtiter plates). Wash reaction conditions, percent purity, polypeptide and poly Solutions commonly contain a physiological buffer to prevent nucleotide lengths, and so forth, used in the specification and denaturation of antigens and their cognate antibodies, and to 55 claims, are modified by the term “about, unless otherwise preserve enzyme activity. Buffers such as PBS, Tris-saline, or indicated. Accordingly, the numerical parameters set forth in imidizole-buffered saline at neutral pH are widely used. Spe the specification and claims are approximations that may vary cific buffers are typically selected based on the method of depending upon the desired properties of the present inven detection to be employed in a particular assay. Wash buffers tion. At the very least, and not as an attempt to limit the should also include non-ionic detergents such as TWEEN 60 application of the doctrine of equivalents to the scope of the 20TM, TRITON X-100TM, or the like, at concentrations of claims, each numerical parameter should at least be construed between 0.01% to 0.05% (v/v), in order to disrupt low-affin in light of the number of reported significant digits, applying ity, non-specific interactions between reaction components. ordinary rounding techniques. Nonetheless, the numerical After the blocking step, the wells of the microtiter plate are values set forth in the specific examples are reported as pre washed. The adsorbed antigen then undergoes the primary 65 cisely as possible. Any numerical value, however, inherently antibody incubation, after which it is washed again. Anti contains certain errors from the standard deviation of its body/antigen complexes are then detected using a secondary experimental measurement. US 7,972,785 B2 13 14 It must be noted that, as used herein and in the appended To confirm the establishment of a rat liver fibrosis model, claims, the singular forms “a,” “or, and “the include plural Q-RT-PCR, serum analysis, and histopathological analysis referents and plural referents include singular forms unless were employed. the context clearly dictates otherwise. Thus, for example, Histopathological Examination of DMN-Induced Liver reference to “a subject polypeptide' includes a plurality of 5 Damage such polypeptides, reference to “the agent” includes refer Liver tissues were immediately removed after sacrifice and ence to one or more agents and equivalents thereof known to subject to histopathological examination. The fixed liver those skilled in the art, reference to “nucleic acid molecules' samples were then processed for paraffin embedding. Five includes reference to one or more nucleic acid molecules, and micrometer (5 um) sections were prepared for hematoxylin 10 and eosin staining (to score necroinflammatory and fatty reference to “antibodies' includes reference to one or more changes) and for Sirius red/fast green collagen staining (to antibodies and so forth. score for fibrosis) (23). The fatty changes were classified as The following examples further illustrate the invention. presence (+) or absence (-). They are merely illustrative of the invention and disclose Phenotypic changes resulting from DMN-induced liver various beneficial properties of certain embodiments of the 15 damage are shown in FIG. 2A. In FIG. 2A, the representative invention. The following examples should not be construed as phenotypes of the DMN-induced rat liver fibrosis were char limiting the invention. acterized by scoring the four histopathological features (ne crosis, inflammation, fibrosis, and fatty change) as follows: EXAMPLES the necrosis scores were from NO to N3 (the first panel), and the inflammation scores were from IO to I3. The scoring The practice of the present invention will employ, unless system for examining the intensity of liver fibrosis was modi otherwise indicated, conventional techniques for the study of fied from the scoring system of the Hepatitis Activity Index differential gene expression, differential proteomic expres (HAI) (29, 30). Fibrosis was divided into four scores: normal sion, and histopathology, which are within the level of ordi (FO), fibrous expansion of portal tracts (F1), bridging fibrosis nary skill in the art. Such techniques are explained fully in the 25 (F2) and frequent bridging fibrosis with focal nodule forma applicable literature. tion (F3). The fatty change was scored as present (+) or The following examples illustrate the study of the expres absent (-), respectively (the last panel). The images of the sion of different proteins in liver fibrosis through the use of fatty change are shown at 200x magnification, whereas the animal models. others are shown at 100x magnification (FIG. 2A). 30 FIG. 2B shows a summary of histopathological scores for Example 1 the rat model. The results were ranked by time course. The necroinflammatory scores were the sum of the necrosis and Establishment of the Rat Hepatic Fibrosis Model inflammation scores and range from A0 to A6. The necroin flammatory change was divided into three grades: A0-none, A rathepatic fibrosis model was set up through the use of 35 A(1-3)-mild and A(4-6)-moderate necroinflammation. The DMN. DMN is a potent hepatoxin that specifically targets the fibrosis change was divided into two grades: F(0-1)-normal liver and can cause liver fibrosis. The histopathological to fibrous expansion of portal tracts, F(2-3)=bridge fibrosis to changes resulting from DMN treatment involve the rapid frequent bridging fibrosis with nodule formation. The fatty deposition of collagen, the major protein of fibrosis and change was shown as presence (+) or absence (-). The num 40 ber of rats was counted and used to calculate the percentage of important to the process of cirrhosis. DMN-induced liver each histopathological level at each time point. injury in rats displays many features, such as portal hyperten As shown in FIG. 2B, seventy-five percent of the DMN sion ascites as well as a number of other histopathological and treated rats had none (FO) or low levels of fibrosis (F1) in the biochemical abnormalities. Previous study has suggested that first two weeks. By the third to fourth week, nearly 90% of the DMN-induced liver injury in rats could reflect changes that 45 DMN-treated rats had high levels of fibrosis, from bridging occur in human hepatic fibrosis and/or cirrhosis and that it is fibrosis (F2) to frequent bridging fibrosis with focal nodule an appropriate animal model for studying human hepatic formation (F3). In the last two weeks, F2 and F3 were still fibrosis and/or cirrhosis (15). The DMN-induced liver fibro present in 78% of DMN-treated rats. The fatty changes were sis model was performed as described in Jezequel A. M. et al. only present in a few treated rats (3.7%). In contrast, there (14). Male Sprague-Dawley rats (Slc:SD: Japan SLC, Shi 50 were no abnormal pathological patterns present in the control Zuoka, Japan), weighing from 300 to 350 grams, were used in group at all. In addition, no clear abnormality was found in the the experiments. To induce hepatic fibrosis over a six-week kidney or spleen of the DMN-treated and normal rats (data time course experiment, the rats were given DMN (Sigma, not shown). Together, the detailed necroinflammatory and Saint Louis, Mo.), dissolved in normal saline, three consecu fibrosis scoring systems of the process of the DMN-induced tive days a week for the first three consecutive weeks at the 55 liver damage Suggest that dramatic necrosis and inflammation dosage of 6.7 mg/kg per body weight by intraperitoneal injec took place during early liver damage progression (week 1-4), tion. Injection time points are shown as inverted triangles in followed by fibrosis formation at 3-6 weeks. Collagen fiber FIG. 1. Injections were at a much lower dosage than those deposition in rat liver could be observed, along with bile duct used in other experiments where the dosage was 100 mg/kg/ proliferation, centrilobular necrosis, bridge fibrosis and fibro day. The higher dosage can cause toxicity in rat liver (21, 22).) 60 sis surrounding the central veins, after three weeks of DMN Two to seven rats at each time point were treated with either treatment. DMN or with an equal volume of normal saline without DMN Quantitative Real-Time Polymerase Chain Reaction as the control (26 DMN-treated rats and 24 control rats). Rats (Q-RT-PCR) were weighed and sacrificed on days 11, 18, 25,32, 39, and 46 To gain additional information about the established ani (sacrifice time points are shown as triangles in FIG. 1A) 65 mal model, Q-RT-PCR was used to evaluate the gene expres respectively. These triangular time points were designated as sion profile of transforming growth factor-1 (TgfB1), which is weeks 1 through 6 (FIG. 1A). the strongest known inducer of fibrogenesis in the effector US 7,972,785 B2 15 16 cells of hepatic fibrosis that can stimulate the adipocyte trans call according to the perfect match (PM)/mismatch (MM) formation (24-27). The same total RNA samples were used algorithm of Affymetrix in all gene chips (28). Of the 8799 for both microarray and Q-RT-PCR analyses. RNA prepara probe sets analyzed, overall expression patterns for 2385 tion and analysis were performed according to Affymetrix's transcripts on the chips were reported to be present (P<0.05). (Santa Clara, Calif., U.S.A.) instructions. The TAOMANR To verify that intra-sample variability did not obscure differ assays were conducted in triplicate for each sample, and a ences between the controls and DMN-treated groups, as well mean value was used for calculation of expression levels. To as to determine the fold-change that should be considered as standardize the quantification of the target genes, 18S ribo significant, expression profiles among the 24 control datasets somal RNA (18S rRNA) from each sample was quantified on were compared. Scattered graphs of expression levels of the the same plate with the target genes. The Q-RT-PCR result 10 2385 transcripts represented on the microarray were com showed that a higher level of mRNA expression of TgfB1 was pared with each other. The relationship between the experi observed in DMN-treated rat livers than in the controls (FIG. mental chips in the microarray analysis was analyzed by 1B). The changes were in agreement with observations linear regression. Overall, there was no statistical difference, described in Jezequel A. M. et al. (14). In Sum, these exami with 3.2% of transcripts deviating more than 2-fold. To inves nations support the DMN-induced rathepatic fibrosis model. 15 tigate the time course variability, the reliable signals of these Analysis of Serum Biochemical Data from the DMN-In 2385 probe sets between the first and sixth week of controls, duced Hepatic Fibrosis Rat Model Experiment were calculated. Again, there was no statistical difference, The serum of each rat, 50 rats in total, was subjected to with 4.6% of transcripts deviating more than 2-fold. In con various biochemical examinations related to liver damages. trast, a significant scatter was found between controls and Blood samples, collected from the animals at necropsy, were DMN-treated groups, with 28.7% of transcripts deviating used to measure serum concentrations or activity of albumin, more than 2-fold. glutamic oxaloacetic transaminase (GOT), glutamic pyruvic As a first step to minimize the likelihood of false positives, transaminase (GPT), total bilirubin, alkaline phosphatase all transcripts were filtered by forming two independent clus (AKP), acid phosphatases (ACP), C.-fetoprotein (AFP), blood ters from the microarray data and identifying those that were urea nitrogen (BUN), cholesterol (CHOL), lactate dehydro 25 differentially expressed. For detailed analysis, the first cluster genase (LDH), globulin, prothrombin time (PT) and blood generated 2385 transcripts as previously described. Of these, platelets (PLT) using an Hitachi 747 and ACL 3000 clinical 268 were differentially expressed transcripts either higher or chemistry analyzer system (MYCO, Renton, Wash.). When lower by 1.5 fold or more when comparing the controls and the DMN-treated samples and controls were compared, thir DMN-treated groups. The second method, which used the teen serum markers showed significant differences and con 30 “detection flag” selection (24), reported 23 transcripts as firmed the DMN-treated group had suffered liver damage. “present in the DMN-treated groups but not in the controls. The biochemical data for the DMN-treated group suggest that In contrast, there was only one transcript reported “absent” in there were changes in many serum markers and that the all DMN-treated groups but “present in the controls. protein expression levels or physical responses are similar to Altogether, 292 transcripts representing 256 genes, includ human liver damage phenotypes (1, 2). The thirteen serum 35 ing 137 up-regulated and 119 down-regulated genes, exhib markers identified in these experiments are the same serum ited differentially expressed gene expression patterns when markers for human liver fibrosis. the DMN-treated groups and controls were compared. Hier archical clustering generated a dendrogram for the gene Example 2 expression patterns of these 292 transcripts across the 24 40 samples as shown in FIG. 3A. The rows represent individual Establishment of Gene Expression Profile During transcripts and columns represent time course samples. The DMN-Induced Liver Fibrosis color in each cell reflects the expression level of the corre sponding tissue, relative to its mean expression level. The Microarray Analysis scale extends from fluorescence radios of 0.25 to 4 relative to The quality of the total RNA for microarray analysis was 45 the mean level for all samples. determined using Spectra Max Plus (Molecular Devices, Quantitative-Real-Time-Reverse-Transcriptase Poly Sunnyvale, Calif., U.S.A.) and had an Ago/Aso ratio ranging merase Chain Reaction (O-RT-PCR) from 1.9 to 2.1. Protocols and reagents for hybridization, To validate the microarray data, Q-RT-PCR analysis was washing and staining followed Affymetrix’s instructions, performed for tissue inhibitor of metalloproteinase 1 which may be retrieved from Affymetrix's company website. 50 (Timp1), tissue inhibitor of metalloproteinase 2 (Timp2), Labeled cRNA was hybridized to the Affymetrix GeneChip matrix metalloproteinase 3 (Mmp3) and gamma-glutamyl Test 3 Array to verify the quality prior to hybridization to the transpeptidase (Ggtp). These genes were chosen for valida Affymetrix Rat Genome U34A Array. tion because these genes occurred both in this GeneChip Data Analysis and Clustering Algorithm study and in previous studies. The microarray images were transformed into text files 55 As determined by Q-RT-PCR, Timp1 (FIG. 3B), Timp2, containing intensity information using GENECHIPR) Oper Mmp3, Ggtp (data not shown) and TgfB1 (shown previously ating Software (GCOS) developed by R Affymetrix. The in FIG. 1B) were elevated in DMN-treated samples. The microarray datasets were then analyzed using GENE results of Q-RT-PCR analysis of these five genes were con SPRING(R) 7.2 software (Silicon Genetics, Redwood City, sistent with previous reports examining these individual Calif., U.S.A.) 60 markers (8, 9). Gene Expression Profiling Moreover, a good concordance based on the fold changes Over the six week time course of the experiment, the liver between microarray data and Q-RT-PCR results was tissues of 12 control animals and 12 DMN-treated rats (2 rats observed. As shown in FIG. 3B, Timp1 expression was for each time point) were selected to perform microarray elevated over 20-fold in DMN-treated rats in both microarray experiments. Before any statistical analyses were applied to 65 and Q-RT-PCR experiments. TAOMANR) assays were con the microarray data, reproducibility was assessed. Genes ducted in triplicate for each sample, and a mean value was were selected as present when they were assigned a present used for calculation of expression levels (marked by the US 7,972,785 B2 17 18 square). To standardize the quantification of the Timp1 tran nucleotide microarray analysis is a powerful approach for script, 18S rRNA from each sample was quantified on the monitoring molecular events during liver injury and repair same plate as the target gene, as indicated by the log scale on where the pathogenesis is unknown. Also, the signature genes the right side of plot. The expression levels of the two Timp1 identified in this study could discriminate successfully transcripts, rc AI169327 at and rc AI 169327 g at (marked 5 between the low-score and the high-score histopathology by a circle and a triangle, respectively), were set forth relative groups. Together, the genes would seem to be responsible for to the mean of all gene expression levels as indicated by the fibrosis formation and are possible markers for the detection scale on the left side of plot. The expression pattern of Timp1 of fibrosis. was highly correlated between the Q-RT-PCR results and the GeneChip analysis (the Pearson's correlation coefficients 10 Example 3 were 0.79 and 0.92, respectively) (FIG. 3B), suggesting that the gene expression results were reliable when subject to Protein Expression Profiling During DMN-Induced more detailed analysis. Liver Fibrosis Fibrosis Candidate Genes Necroinflammatory and fibrosis have been suggested to 15 Experiments using iTRAO tagging were performed to play important roles in the progression of liver cirrhosis in the identify differentially expressed proteins in liver fibrosis. rat model (8,23, 24, 29, 30). To clarify the factors responsible Isobaric Tagging Method: Sample Preparation and for the histopathological phenotype, all rat samples were Reagent Labeling classified by histopathological evaluation with histopatho The liver tissues of 6 control and 6 DMN-treated rats were logical scores for necroinflammation (AO-A6) and fibrosis 20 used to perform duplicate iTRAO labeling experiments. Liver (F0-F3) as described earlier in FIG. 2B. The student t-test tissue was frozen in liquid nitrogen and then pulverized to statistic analysis was used for fibrosis-related genes analysis powder using a mortar and pestle pre-cooled with liquid as it was based on the two-subgroup (F(0-1) and F(2-3)) nitrogen. Then, the liquid nitrogen was allowed to evaporate, variation in fibrosis score. A P-value of less than 0.05 was and the powdered tissue was separated into tubes and stored at considered to be statistically significant. 25 -80°C. Twenty volumes (w/v) of lysis buffer (containing 2% Using the student's t-test, the inventors analyzed those 256 (w/v) SDS in 20 mM phosphate buffer at pH 7.6) was added genes with expression either higher or lower by 1.5 fold or to the powdered tissue and incubated at room temperature for more in DMN-induced rats than in control rats. A total of 62 1 hour. Samples were centrifuged at 12000 rpm for 10 min differentially expressed genes (32 up regulated and 30 down utes and the Supernatant was taken for acetone precipitation regulated) between the F(0-1) and F(2-3) level of fibrosis 30 by adding six volumes of cold acetone to the sample tubes. were identified, estimated using only two subgroup variations Tubes were incubated at -20° C. until precipitate formed. for the fibrosis score, at the 5% significance level. In agree After decanting the acetone, pellets were resuspended with ment with previous studies, three genes, including Timpl. 0.1% SDS and 6M urea in dissolution buffer (provided by CD63 and annexin A1 (Anxa1), exhibited similar gene iTRAQ Reagents Kit, Applied Biosystems, Foster City, expression patterns during liver fibrosis (31-34). 35 Calif., U.S.A.). Timp1 is a well-known fibrosis marker and has been The total protein contents were determined using the Coo proven to play a significant role in the progression of liver massie Plus Protein Assay Reagent (PIERCE, Rockford, Ill., fibrosis. In the later stages of liver injury, hepatic stellate cells U.S.A.). Two hundred (200) ug of proteins were taken and (HSCs) express a combination of matrix metalloproteinases diluted by an equal volume (w/v) of dissolution buffer con (MMPs) that have the ability to degrade normal liver matrix, 40 taining 0.1% (w/v) SDS. Proteins were reduced and cysteines while inhibiting degradation of the fibrillar collagens that blocked as described in the iTRAO protocol (Applied Bio accumulate in liver fibrosis. An increase in expression of systems, Foster City, Calif., U.S.A.). Four volumes of disso Timp1 leads to a more global inhibition of degradation of lution buffer were added to each tube and incubated with 20 fibrillar liver collagens by interstitial collagenases (MMP-1/ ug Trypsin at 37° C. for 16 hours. Tryptic peptides extracted MMP-13) (35). As shown in FIG. 3B, expression of Timp1 45 from liver tissue of controls and DMN-treated rats were was elevated more than twenty fold in DMN treated rats in labeled with iTRAQ 114 and 115, respectively, at each time microarray. point of the experiment. CD63, a transmembrane protein, one of the fibrosis gene Peptide Separation and Analysis signatures observed, is also up-regulated after DMN treat Peptides with different isobaric tags were pooled and acidi ment. Following chronic injury, HSCs activate or differenti- 50 fied by mixing with 10 mM phosphoric acid (in 25% (v/v) ate into myofibroblast-like cells, acquiring contractile, proin acetonitrile and 75% H. Sub.2O) to a total volume of 4.0 mL flammatory, and fibrogenic properties. Activated HSCs for strong cation exchange (SCX) chromatography. The migrate and accumulate at the sites of tissue repair, secreting resulting sample was injected into a liquid chromatography large amounts of extracellular matrix proteins during the pro system (Ettan, GE Healthcare Bio-Science, Umea, Sweden) gression of fibrosis. Activated HSCs have been identified as 55 using a 2.1 mm.x200 mm Polysulfoethyl A column packed major collagen-producing cells (collagen is an extracellular with 5 micron 300A beads (PolyLC, Columbia, Md., U.S.A.) matrix protein) and an initiator of liver fibrosis when the liver at a flow rate of 0.08 ml/min. A guard column of the same is injured (7). It has been demonstrated that inhibition of material was plumbed upstream from the analytical column. CD63 might be a novel diagnostic marker for the injured liver. The buffers used were 10 mM. KHPO 25% (v/v) acetoni Annexin A1 (Anxal) is highly expressed after liver injury 60 trile at pH3.0 for buffer A and 10 mMKHPO, 1MKC1, 25% in an alcoholic liver disease (ALD) study (34). Alcohol initi (v/v) acetonitrile at pH 3.0 for buffer B. The elution gradient ated liver injury occurs via inflammation. ALD progression was changed linearly from 0 to 40% buffer B within 16 mL involves continuing liver injury, fibrosis, and impaired liver and then up to 100% buffer B in another 4 mL. A total of 30 regeneration. It has been Suggested that Anxal might play a fractions were collected and dried by speed vacuuming cen role in the progression of fibrosis. 65 trifugation. All the fractions were desalted on PEPCLEANTM The fact that the present study agrees with previous studies C-18 spin columns (PIERCE, Rockford, Ill., U.S.A.). The for the three genes as described above indicated that oligo desalted peptide from each SCX fraction was dried by speed US 7,972,785 B2 19 20 vacuuming centrifugation and then analyzed by nanoLC In addition, glycine N-methyltransferase (Gnmt), aldolase hybrid mass spectrometry. The nanoLC system was from LC A(Aldoa), Myosin light polypeptide 6 (Mylo) and, cytoplas Packings (Amsterdam, The Netherlands), coupled to an API mic Y actin, (Acty)) were identified in both RNA expression QSTAR Pulsar Hybrid QqTOF mass spectrometer (Applied microarray and proteomic studies as exhibiting differential Biosystems/MDS Sciex, Foster City, Calif., U.S.A.). Peptide expression. separation was performed using a reversed-phase Cs column (75 um in diameterx15 cm in length, 3 um particles) with a Example 4 two-step linear gradient of 5-50% Buffer A over 45 minutes and 50-95% Buffer B over 10 minutes at a flow rate of Network Analysis 0.2.mu.1/minute (Buffer A: 2% (v/v) acetonitrile, 0.1% (w/v) 10 formic acid; Buffer B: 80% (v/v) acetonitrile, 0.1% (w/v) To further refine the genes with differential expression in formic acid). The m/z scanning range for MS and tandem MS the fibrotic liver, we carried out network analysis using the (MS/MS) were 400-1200 and 75-1500 Da, respectively. Ingenuity Pathway Analysis (IPA) software (Ingenuity Sys MS/MS mass spectra were analyzed in continuous flow mode tems, Mountain View, Calif., U.S.A.). The genes, which were with a 10 mm I.D. fused silica tip. MS/MS spectra were 15 identified by microarray and iTRAQ proteomic analyses, analyzed against rat.fasta databases from NCBI by Pro were Subjected to interacting network analyses. Gene acces QUANT 2.0 (Applied Biosystems/MDS Sciex, Foster City, sion numbers were imported into IPA software. These net Calif., U.S.A.). The accuracy tolerance for peptide identifi works described functional relationships between gene prod cation was 0.5 Da for MS and 0.5 Da for MS/MS, respec ucts based on known interactions in the literature. The IPA tively. The cut off for the confidence settings was at 95 and for tool then associated these networks with known biologic the score was at 20. Relative quantification of peptide was pathways. Of sixty-two genes identified from the microarray performed on the MS/MS scans, using ratio of the areas under study, forty-two genes fall into 3 groups, i.e., gene networks the peaks. Relative quantification of protein was obtained by associated with 1) cancer, cell morphology, and dermatologi averaging the constituted peptides identified. cal diseases and conditions; 2) lipid metabolism, Small mol To validate the method and results obtained from iTRAQ 25 ecule biochemistry, molecular transport; and 3) cancer, cel proteomic studies, the expression changes of Vimentin quan lular movement, cellular growth and profileration. See Table tified by mass spectrometry in iTRAO experiments were 3A. Twenty-two out of thirty-nine proteins identified in the compared with those obtained through Western blot with proteomic study fall into another 3 networks associated with anti-vimentin antibody. Proteins were extracted with 2% 1) cancer, cell cycle and cell morphology; 2) lipid metabo (w/v) SDS in 20 mM phosphate buffer at pH 7.6 from control 30 lism, Small molecule biochemistry, organismal injury and and DMN-induced rat livers. After resolving by 12% SDS abnormalities; and 3) hematological disease, endocrine sys PAGE, proteins were transferred to polyvinylidene difluoride tem development and function, nervous system development membranes and Subsequently immunostained with mouse and function. See Table 3B. Table 3A and 3B list the net anti-vimentin monoclonal antibody (Chemicon, Temecula, works protein functions and members. Genes identified in Calif., U.S.A.) for 1 hour, then with horseradish peroxidase 35 the present study are underlined. The underlined genes that labeled goat anti-rat IgG for another hour. The immunoreac have not been reported to associate with liver injury are shown tive bands were detected using an enhanced chemilumines in bold. cence system (ECL, Perkin-Elmer Life Sciences, Wellesley, Mass., U.S.A.). As demonstrated in FIG. 4, the results Example 5 observed in Western blots matched the trend as measured by 40 mass spectrometry. Clinical Validation of Biomarkers Identified in the Results DMN-Induced Liver Fibrosis Rat Model with The numbers of distinct peptides identified range from Human Samples 624-1591 when the confidence threshold was set at 95%. A significant number of these peptides were identified more 45 To confirm that the biomarkers identified in the DMN than once. More than 351 unique proteins were identified in induced liver fibrosis rat model could be used to diagnose each experiment. Thirty-nine proteins show significant and liver fibrosis or cirrhosis in humans, we examined expression consistent differences (1.5 fold or more) in protein expression of the enzyme carbonic anhydrase I (see Table 2, SEQ ID in DMN-treated liver tissue with F2 and F3 fibrosis score NOS: 11 and 71) in human sera by Western blot analysis. compared to control rats. Among these 39 proteins, several 50 Serum samples (8 y1 from each patient) from normal volun have previously been reported to associate with fibrosis for teers and from patients suffering from hepatitis B-related mation, including, fibrinogen, and fibronectin. cirrhosis were first treated to remove the twenty high abun Fibrinogen was found to be up-regulated in DMN-induced dance serum proteins using the PROTEOPREPR 20 Plasma rats in the present study. This is consistent with the results of Immunodepletion Kit (Sigma-Aldrich, St. Louis, Mo., fibrinogen gene expression in a model CC1-induced rat liver 55 U.S.A.). After depletion, the low abundance serum proteins damage. The CC1-induced rat liver damage also showed were precipitated with 5 volumes of cold acetone at -20°C. increased fibrinogen mRNA levels and fibrinogen/fibrin for 2 hours. The protein/acetone mixture was then centrifuged deposition during short-term liver injury and liver fibrogen at 12,000 rpm for 10 minutes. The protein pellet was resus esis. This may suggest that fibrinogen involves a "clotting pended, separated by electrophoresis on a 12% SDS-PAGE like process” in short term liver damage and liver fibrosis 60 gel, and transferred to polyvinylidene difluoride (PVDF) (36). Fibronectin consists of two polypeptide chains. It medi membranes (Millipore, Billerica, Mass., U.S.A.). After ates adhesion of collagen, fibrin and heparin to cells and is blocking in 5% (w/v) nonfat milk, the membranes were thus involved in the organization of thrombi and in wound washed with TBST buffer (20 mM Tris-HCl, pH 7.6, 137 mM healing by inducing attachment of these structures to cells. NaCl, 0.1% (v/v) TWEEN-20) and incubated with goat poly Fibronectin and collagen types I and III have been used as a 65 clonal antibody specific for carbonic anhydrase I (Abcam, characteristic feature of a liver cirrhotic state in the CC1 Cambridge, UK) for 1 hour. The membranes were washed induced liver cirrhosis rat model (37). again with TBST buffer and immunostained with horseradish US 7,972,785 B2 21 22 peroxidase-conjugated donkey anti-goat IgG (Jackson 15. George, J., Rao, K. R., Stern, R., and Chandrakasan, G. ImmunoResearch Laboratories, Pa., U.S.A.). The immunore Dimethylnitrosamine-induced liver injury in rats: the early active bands were visualized using an enhanced chemilumi deposition of collagen, Toxicol. 156: 129-138 (2001). nescence system (ECL, Perkin-Elmer Life Sciences, Welles 16. DeSouza, L., Diehl, G., Rodrigues, M.J., Guo, J., Rom ley, Mass., U.S.A.). The Western blots clearly show that some 5 aschin, A. D., Colgan, T. J., and Siu, K. W. Search for serum samples from the cirrhosis patients have higher con cancer markers from endometrial tissues using differen centrations of carbonic anhydrase I than samples from normal tially labeled tags iTRAO and cICAT with multidimen individuals not suffering from cirrhosis. 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M. “Hybridization With Radioactive Probes.” 36. Neubauer, K., Knittel, T., Armbrust, T., and Ramadori, G. in Current Protocols in Molecular Biology 6.3.1-6.3.6. Accumulation and cellular localization offibrinogen/fibrin (John Wiley & Sons, N.Y. 2000). during short-term and long-term rat liver injury, Gastroen 41. SAMBROOK ET AL, 3 MOLECULAR CLONING: A LABORATORY terol. 108(4): 1124-35 (1995). MANUAL A8.40-A8.45, and A8.52-A8.55 (2001). TABLE 1 Clinical, chemical and fibrosis parameters in treated and untreated groups of rats Numeric control DMN-treatment variable 1-2 wk (n) 3-4 wk (n) 5-6 wk (n) 1-2 wk (n) 3-4 wk (n) 5-6 wk (n) Albumin (g/dl) 4.4 + 0.4 (7) 4.6+ 0.2 (8) 4.7 + 0.2 (8) 3.9 + 0.7 (7) 3.5 + 0.6 (11) 3.2 + 0.1 (7) GPT (U/1) 61.1 + 26.7 (8) 65.9 + 19.7 (7) 50.3 + 4.9 (8) 459.5 + 78.5 (8) 566.6+ 313.5 (11) 763.6+ 405.2 (7) GOT (U/1) 110.3 + 37.6 (8) 84.0 + 23.5 (7) 109.1 + 23.5 (8) 661.5 + 1344 (8) 1006.1 + 749.6 (11) 1572.9 + 965.3 (7) Bilirubin 0.13 + 0.05 (8) 0.10 + 0.01 (8) 0.13 + 0.05 (8) 0.72 + 0.53 (8) 1.01 + 0.74 (11) 1.13 + 1.00 (7) (mg/dl) AKP (KA) 46.0 + 3.7 (4) 44.8 + 2.2 (4) 47.0 + 13.6 (4) 600.8 + 93.0 (4) 668.3 + 222.0 (3) 468 + 12.7 (2) LDH (IU/I) 262.3 + 75.1 (4) 289.3 + 31.7 (3) 292.3 + 31.3 (4) 414.8 + 102.7 (4) 562.0 + 120.8 (3) 853.5 + 91.2 (2) Globulin (g/dl) 6.9 + 0.3 (3) 6.9 + 0.5 (4) 7.3 + 0.2 (4) 6.7 + 0.1 (2) 5.0 + 0.8 (4) 3.6 + 0.3 (2) AFP (ngfdl) 0.32 + 0.04 (4) 0.2 + 0.01 (2) 0.24 + 0.03 (4) 0.40 + 0.19 (4) 0.38 + 0.05 (4) 0.35 + 0.07 (2) CHOL (mg/dl) 88 + 5 (4) 71 + 20 (4) 91 + 5 (4) 77 + 8 (4) 70 + 13 (6) 67 + 18 (5) BUN (mg/dl) 31 + 2 (4) 25 + 6 (4) 26 + 9 (4) 33 + 4 (4) 36 + 2 (4) 31 + 5 (2) ACP (mg/dl) 2.3 + 0.8 (4) 2.6+ 0.5 (4) 2.3 + 0.8 (4) 1.9 + 0.6 (4) 6.2 + 1.1 (4) 8.2 + 0.6 (2) PT (sec) 14 + 1 (7) 13 + 1 (8) 13 + 1 (7) 18 + 4 (8) 20 + 4 (9) 22 + 5 (6) PLT (10/-1) 741 + 245 (8) 981 + 124 (8) 893 + 109 (8) 407 + 72 (7) 300+ 165 (11) 229 + 302 (7) GPT, glutamic pyruvic transaminase; GOT, glutamic oxaloacetic transaminase; ilirubin, total bilirubin; KP, alkaline phosphatase;

L. DH, lactate dehydrogenase; FP, a-fetoprotein; HOL, cholesterol; UN, blood urea nitrogen; CP, acid phosphatase; A prothrombin time; LT, blood platelets. Mean SD of value from 1-2, 3-4 or 5-6 week in treated and untreated groups,

TABLE 2 The 63 selected genes and their human orthologs

Human Ex Homo pres Gene Human orthologs- Official log ID Method sion Protein name-Rat Official Symbol ID ID l8le Symbol GeneD SEQ Proteomics down cytochrome P450, Cyp2c23 83790 ID family 2, Subfamily NO: 1 c, polypeptide 23 SEQ Proteomics down cytochrome P450, Cyp2d 13 24303 ID family 2, Subfamily NO: 2 d, polypeptide 13 SEQ Proteomics down fatty acid synthase Fasn 50671 SEQ ID fatty acid synthase FASN 2194 ID NO: 64 NO:3 US 7,972,785 B2 25 26 TABLE 2-continued The 63 selected genes and their human Orthologs

Human Ex Homo pres Gene Human orthologs Official log Method sion Protein name-Rat Official Symbol ID ID l8le Symbol GeneD Proteomics down hydroxysteroid (17 79.243 SEQID hydroxysteroid (17 3294 beta) dehydrogenase 2 NO: 65 beta) dehydrogenase 2 4 Proteomics down Estrogen Ste2 SEQID Sulfotransferase, SULT1E1 6783 Sulfotransferase, NO: 66 estrogen-preferring 5 isoform 2 Proteomics down similar to cDNA 362399 SEQID ZXD family Zinc 79364 sequence BC022133 NO: 67 finger C 6 Proteomics S100 calcium S100a 941.95 SEQID S100 calcium S100A9 binding protein A9 NO: 68 binding protein A9 7 (calgranulin B) Proteomics actin, gamma, Actg(Actgl) 287876 cytoplasmic 8 Proteomics aldehyde 24188 SEQID aldehyde ALDH1A1 216 dehydrogenase NO: 69 dehydrogenase 1A1 9 family 1, member A1 Proteomics Rho GDP Arhgdia 360678 SEQID Rho GDP ARHGDIA 396 dissociation inhibitor NO: 70 dissociation inhibitor (GDI) alpha (GDI) alpha Proteomics carbonic anhydrase I Ca1 predicted 310218 SEQID carbonic anhydrase I CA1 759 (predicted) NO: 71

Proteomics carbonic anhydrase 2 Ca2 S4231 SEQID carbonic anhydrase CA2 760 NO: 72 II

S Proteomics cytochrome P450 Cyp2c13 171521 2c13

QO Proteomics thyroid autoantigen SEQID ATP-dependent 2547 NO: 73 DNA helicase II, 70 kDa subunit

Proteomics myosin, heavy 25745 SEQID myosin, heavy 4627 polypeptide 9 NO: 74 polypeptide 9, non muscle

Proteomics Myosin light Mylo SEQID similar to Myosin 124685 polypeptide 6 NO: 75 light polypeptide 6 (Myosin light chain alkali 3) (Myosin light chain 3) (MLC 3) (LC17) Proteomics up prosaposin Psap SEQID prosaposin PSAP S660 NO: 76

Proteomics up similar to Myosin RGD1309537 predicted SEQID myosin regulatory 10627 regulatory light NO: 77 light chain MRCL3 chain 2-A, Smooth muscle isoform (Myosin RLC-A) (predicted) Proteomics up solute carrier family 24779 SEQID solute carrier family 6521 4, member 1 NO: 78 4, anion exchanger, member 1 (erythrocyte membrane protein band 3, Diego blood group) SEQ Proteomics up transgelin 2 Taglin2 SEQID transgelin 2 TAGLN2 ID (predicted) NO: 79 NO: 2O US 7,972,785 B2 27 28 TABLE 2-continued The 63 selected genes and their human Orthologs

Human Ex Homo pres Gene Human orthologs Official log Method sion Protein name-Rat Official Symbol ID ID l8le Symbol GeneD Proteomics up tropomyosin 3, Tpm3 117557 SEQID tropomyosin 3 TPM3 7170 gamma NO: 80

Proteomics up talin TIn 313494 SEQID talin 1 TLN1 7094 NO: 8

Proteomics up similar to RGD1308525 predicted 298381 SEQID enoyl Coenzyme A ECHDC2 55268 hypothetical protein NO: 82 hydratase domain D4Ertf65e containing 2 (predicted) Microarray OW aminoadipate Aadat 29416 SEQID alpha-aminoadipate AADAT S1166 aminotransferase NO: 83 aminotransferase

Microarray OW acyl-CoA synthetase AcSI1 25288 SEQID acyl-CoA synthetase ACSL1 218O long-chain family NO: 84 long-chain family NO member 1 member 1

Microarray OW aldehyde Ftf 64392 SEQID aldehyde 10840 dehydrogenase 1 NO: 85 dehydrogenase 1 family, member L1 family, member L1

Microarray OW arginosuccinate ASS 25698 SEQID argininosuccinate ASS 445 synthetase NO: 86 synthetase

Microarray OW dimethylglycine Dmgdh 245961 SEQID dimethylglycine DMGDH 29958 dehydrogenase NO: 87 dehydrogenase NO

Microarray OW dihydropyrimidinase Dpys 65135 SEQID dihydropyrimidinase DPYS 1807 NO: 88

Microarray OW phosphodiesterase Enpp2 84050 SEQID ectonucleotide ENPP2 S168 If nucleotide NO: 89 pyrophosphatase? pyrophosphatase 2 phosphodiesterase 2 (autotaxin) Microarray OW fatty acid amide Faah 29347 SEQID fatty acid amide FAAH 21 66 hydrolase NO: 90 hydrolase O:

Microarray OW guanidinoacetate Gamt 25257 SEOID guanidinoacetate N GAMT 2593 methyltransferase NO: 9 methyltransferase

Microarray OW L-gulono-gamma Gulo 60671 - actone oxidase

Microarray OW 3 Hibadh 63938 SEQID 3 HIBADH 11112 hydroxyisobutyrate NO: 92 hydroxyisobutyrate dehydrogenase dehydrogenase

Microarray OW ketohexokinase Khk 25659 SEQID ketohexokinase KHK 3795 NO: 93 s Microarray OW kynureninase (L- Kynu 116682 SEQID kynureninase (L- KYNU 8942 kynurenine NO: 94 kynurenine NO hydrolase) hydrolase) s Q: Microarray OW Pyruvate PC 25104 SEQID pyruvate carboxylase PC SO91 carboxylase NO: 95 NO S Microarray OW pyruvate Pdk2 81530 SEQID pyruvate PDK2 S164 f dehydrogenase NO: 96 dehydrogenase kinase 2 subunit p45 kinase, isoenzyme 2 No (PDK2) US 7,972,785 B2 29 30 TABLE 2-continued The 63 selected genes and their human Orthologs

Human Ex Homo pres Gene Human orthologs Official log Method sion Protein name-Rat Official Symbol ID l8le Symbol GeneD SED Q Microarray down solute carrier family Slc 10a1 24777 solute carrier family SLC10A1 6554 10, member 1 O (sodium/bile acid cotransporter amily), member 1 Microarray down solute carrier family 29573 solute carrier family tD 37 (glycerol-6- 37 (glycerol-6- phosphate phosphate transporter), member 4 ransporter), member 4 Microarray down similar to SO1086 sulfotransferase SULT1C1 6819 Sulfotransferase K2 ggQ amily, cytosolic, (rSULT1C2A) C, member 1 Microarray arachidonate 5 Alox5ap 29624 D arachidonate 5 ALOXSAP 241 lipoxygenase OO ipoxygenase activating protein activating protein

Microarray adenine Aprt predicted adenine APRT 353 phosphoribosyl phosphoribosyl NO transferase ransferase (predicted) s Microarray brain abundant, Basp1 64-160 brain abundant, BASP1 104.09 , membrane attached O2 membrane attached signal protein 1 signal protein 1 Ri. Microarray benzodiazepin 24230 peripheral 706 receptor benzodiazapine receptor R Microarray carbonyl reductase Cbr1 29224 carbonyl reductase 1 CBR1 873 NO

Microarray CD47 antigen (Rh Cd47 29364 CD47 antigen CD47 961 related antigen, integrin-associated signal transducer) Microarray embigin Emb 114511 embigin homolog EMB 1334.18

Microarray myeloid Myd116 171071 protein phosphatase PPP1R15A 23645 differentiation 1, regulatory Subunit primary response 15A gene 116 Microarray N-myc downstream Ndrg2 171114 E N-myc downstream NDRG2 57447 t regulated gene 2 s regulated gene 2 5 O s Eo Microarray expressed in non Nme1 191575 non-metastatic cells NME1 4830 metastatic cells 1, EEEEEEEEEEE 1, protein (NM23A) N. :51 protein (NM23A) sSSSSSSSSSS expressed in (nucleoside diphosphate kinase) Microarray neuregulin 1 1124OO s E neuregulin 1 NRG1

Microarray oxidized low density Oldr1 140914 oxidised low density OLR1 4973 lipoprotein (lectin lipoprotein (lectin like) receptor 1 like) receptor 1 Microarray protein tyrosine 315265 gg twinfilin-like protein 11344 kinase 9 OENOSE

Microarray Syndecan binding Sdcbp 83841 E Syntenin SDCBP 6386 protein s NO

Microarray serine protease Spink1 24833 serine protease SPINK1 6690 inhibitor, Kazal type 1 inhibitor, Kazal type 1 No: US 7,972,785 B2 31 32 TABLE 2-continued The 63 selected genes and their human Orthologs

Human Ex- Homo pres- Gene Human orthologs- Official log ID Method sion Protein name-Rat Official Symbol ID ID l8le Symbol GeneD SEQ Microarray up tyrosine Ywhah 25576 SEQID tyrosine YWHAH 7533 ID 3/tryptophan 5- NO: 115 3/tryptophan 5 NO: monooxygenase monooxygenase 57 activation protein, activation protein, eta polypeptide eta polypeptide SEQ Microarray up S100 calcium S100a10 81778 SEQID S100 calcium- S100A10 6281 D binding protein A10 NO: 116 binding protein A10 NO: 58 SEQ Microarray up B-cell translocation Btg2 296.19 SEQID B-cell translocation BTG2 7832 D gene 2 NO: 117 gene 2 NO: 59 SEQ Microarray up 3'-phosphoadenosine PapSS2 predicted 2941.03 SEQID 3'-phosphoadenosine PAPSS1 9061 D 5'-phosphosulfate NO: 118 5'-phosphosulfate NO: synthase 2 synthase 1 60 (predicted) SEQ Microarray up biogenesis of Bloc1s2 293938 SEQID biogenesis of BLOC1S2 282991 D lysosome-related NO: 119 lysosome-related NO: organelles complex- organelles complex 6 1, Subunit 2 1, Subunit 2 SEQ Microarray up Similar to RIKEN RGD1310681 predicted 297903 D cDNA 672O467CO3 NO: (predicted) 62 SEQ Microarray up transformed mouse Mdm2 predicted 314856 SEQID mouse double MDM2 4193 D 3T3 cell double NO: 120 minute 2 homolog NO: minute 2 (predicted) 63

TABLE 3A Interacting network analysis of proteins selected from microarray study Genes Top functions 1 ACTG1 ADARB1, AHCY, AKT1, ALDOA, BASP1, BZRP, CD63, Crisp1 Cancer, Cell Morphology, (MGI: 102553), EMB, FAAH, GLI1, GNMT, HRAS, IFNG, KHK, KRT18, MYC, MYCN, Dermatological Diseases and Conditions NDRG2, NME1, OTC, PPARA, RHOB, RPL23, RPL27, RPL35, RPL38, RPL41, Rpm12, RPS16, RPS27, RT1-A, SARDH, SLC37A4 2 ACSL1, ALOXSAP, ANXA1, ASS, ATF3, CD47, CIDEC, CP, DF, FABP1, GPD1, Lipid Metabolism, Small Molecule HOXA11, KYNU, MMP16, MTP,MYL6, OLR1, PC, PDK2, PF4. PLIN, PLP1, PPARG, Biochemistry, Molecular Transport PPP1R15A, PROC, PTGES, RGS7, SLC27A1, SPHK1, TIMP1, TNF, UBQLN2, UCP2, UCP3, Wap 3 ACO2, AKR1B10, BTG2, CBR1, DAD1, DUSP5, ELF3, ENPP2, FGF2, GBP1, GLG1, Cancer, Cellular Movement, Cellular GPI, GRIN2C, HSPA1A, IGFALS, IL1B, JUN, KCNH2, MAOB, MT1L, MT2A, NR3C1, Growth and Proliferation NRG1, P53AIP1, PHKG1, S100A10, SDCBP, SHBG, SLC10A1 SLC10A2, SPINK1, SPRY1, TP53, YWHAH, ZFP36L2 *Genes identified in the present study are underlined. The underlined genes that have not been reported to associate with liver injury are in bold.

TABLE 3B Interacting network analysis of proteins selected from iTRAQ proteomic study Genes Top functions 1 ADD1, ALB, ALDH1A1, CA2, CASP3, CNN2, CTNNB1, DIA1, ENPP1, GBA, Cancer, Cell Cycle, Cell Morphology GM2A, HGF, HSD17B2, IL1B, ITPA, JTV1, KRT17, LMNA, LMNB2, MAP4, MYC, OAT, PLS3, PSAP, S100A9, SERPINH1, SLC4A, SRM, SULT1E1. TAGLN2. TGFB1, TLN1, TPM3, UGDH, VIM 2 ACADM, ACTC, ADCY7, ALDH2, ALDOA, ALDOB, ARG2, ARHGDA, Lipid Metabolism, Small Molecule CD276, CNR2, CPS1, CROT, Cyp4a10, Cyp4a14, EHHADH, EPHX1 FAAH, Biochemistry, Organismal Injury and FASN, FN1. G.22P1, GNMT, GOT2, HOXD4, HPX, HRSP12, Ifill, IFNG, IL4, Abnormalities LEP, Mug1, MYH9, MYOD1, PPARA, SORD, TGM2 US 7,972,785 B2 33 34 TABLE 3B-continued Interacting network analysis of proteins selected from iTRAQ proteomic study Genes Top functions 3 ALBANXA1, CLU, CYP2C13, CYP3A2, FGA, FGB, FGG, FLI1, GAL, GFAP, Hematological Disease, Endocrine System GH1, GHRH, GHRL, HBA1, HBA2, HBB, Hbb-ar, Hbb-b1, Hbb-b2, Hbb-bh1, Development and Function, Nervous Hbb-y, HBD, HBE1, HBG1, HBG2, HBQ1, HBZ, IL6, NTS, SOCS2, Stat5, TF, TG, System Development and Function TTR

*Genes identified in the present study are underlined. The underlined genes that have not been reported to associate with liver injury are in bold.

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS : 12 O

<210s, SEQ ID NO 1 &211s LENGTH: 494 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 1 Met Glu Lieu. Leu Gly Phe Thir Thr Lieu Ala Leu Val Val Ser Val Thr 1. 5 1O 15 Cys Lieu. Ser Lieu Lleu Ser Val Trp Thir Lys Lieu. Arg Thr Arg Gly Arg 2O 25 3O Lieu Pro Pro Gly Pro His Pro Pro Ser His Tyr Trp Glu Ser Thr Ala 35 4 O 45 Thr Glu Pro Gln Gly His Pro Ala Ser Lieu. Ser Lys Lieu. Ala Lys Glu. SO 55 6 O Tyr Gly Pro Val Tyr Thr Lieu. Tyr Phe Gly Thr Ser Pro Thr Val Val 65 70 7s 8O Lieu. His Gly Tyr Asp Val Val Lys Glu Ala Lieu. Lieu. Glin Glin Gly Asp 85 90 95 Glu Phe Lieu. Gly Arg Gly Pro Lieu Pro Ile Ile Glu Asp Thr His Lys 1OO 105 11 O Gly Tyr Gly Lieu. Ile Phe Ser Asn Gly Glu Arg Trp Llys Val Met Arg 115 12 O 125 Arg Phe Ser Lieu Met Thr Lieu. Arg Asn. Phe Gly Met Gly Lys Arg Ser 13 O 135 14 O Lieu. Glu Glu Arg Val Glin Glu Glu Ala Trp Cys Lieu Val Glu Glu Lieu. 145 150 155 160 Gln Lys Thr Lys Ala Glin Pro Phe Asp Pro Thr Phe Ile Leu Ala Cys 1.65 17O 17s Ala Pro Cys Asn Val Ile Cys Ser Ile Lieu. Phe Asn Asp Arg Phe Glin 18O 185 19 O Tyr Asn Asp Llys Thr Phe Lieu. Asn Lieu Met Asp Lieu. Lieu. Asn Lys Asn 195 2OO 2O5 Phe Glin Glin Val Asn Ser Val Trp Cys Glin Met Tyr Asn Lieu. Trp Pro 21 O 215 22O Thir Ile Ile Llys Tyr Lieu Pro Gly Llys His Ile Glu Phe Ala Lys Arg 225 23 O 235 24 O Ile Asp Asp Wall Lys Asn. Phe Ile Lieu. Glu Lys Wall Lys Glu. His Glin 245 250 255 Llys Ser Lieu. Asp Pro Ala Asn Pro Arg Asp Tyr Ile Asp Cys Phe Lieu. 26 O 265 27 O Ser Lys Ile Glu Glu Glu Lys Asp Asn Lieu Lys Ser Glu Phe His Lieu. 27s 28O 285 US 7,972,785 B2 35 36 - Continued

Glu Asn Luell Ala Wall Gly Ser Asn Luell Phe Thir Ala Gly Thir Glu 29 O 295 3 OO

Thir Thir Ser Thir Thir Lell Arg Phe Gly Luell Luell Lell Lell Met Lys Tyr 3. OS 310 315

Pro Glu Wall Glin Ala Wall His Glu Glu Luell Asp Arg Wall Ile Gly 3.25 330 335

Arg His Glin Pro Pro Ser Met Asp Lys Met Lell Pro Thir 34 O 345 35. O

Asp Ala Wall Luell His Glu Ile Glin Arg Tyr Ile Thir Lell Wall Ser 355 360 365

Ser Luell Pro His Ala Wall Wall Glin Asp Thir Lys Phe Arg Asp Wall 37 O 375 38O

Ile Pro Gly Thir Thir Wall Luell Pro Met Luell Ser Ser Wall Met Luell 385 390 395 4 OO

Asp Glin Glu Phe Ala Asn Pro Glu Lys Phe Asp Pro Gly His Phe 4 OS 415

Lell Asp Asn Gly Phe Lys Thir Asp Phe Wall Pro Phe 425 43 O

Ser Luell Gly Ala Wall Gly Glu Ser Lell Ala Arg Met Glu 435 44 O 445

Lell Phe Luell Phe Phe Thir Thir Luell Luell Glin Lys Phe Ser Luell Thir 450 45.5 460

Lell Wall Glu Pro Asp Lell Asp Ile Pro Ile Thir Thir Gly Ile 465 470 48O

Ile Asn Luell Pro Pro Pro Luell Cys Luell Wall Pro Arg 485 490

<210s, SEQ ID NO 2 &211s LENGTH: SOO 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 2

Met Glu Lieu. Lieu Ala Gly Thir Gly Luell Trp Pro Met Ala Ile Phe Thir 1. 15

Wall Ile Phe Ile Lell Lell Wall Asp Luell Met His Arg Arg Glin Arg Trp 25 3O

Thir Ser Arg Tyr Pro Pro Gly Pro Wall Pro Trp Pro Wall Luell Gly Asn 35 4 O 45

Lell Luell Glin Wall Asp Lell Cys Asn Met Pro Tyr Ser Met Luell SO 55 6 O

Glin Asn Arg Tyr Gly Asp Wall Phe Ser Luell Glin Met Gly Trp Pro 65 70

Wall Wall Wall Ile Asn Gly Lell Ala Wall Glin Glu Lell Luell Wall Thir 85 90 95

Gly Glu Asp Thir Ala Asp Arg Pro Glu Met Pro Ile Phe Glin His 1OO 105 11 O

Ile Gly Tyr Gly His Ala Lys Gly Wall Wall Lell Ala Pro Gly 115 12 O 125

Pro Glu Trp Arg Glu Glin Arg Arg Phe Ser Wall Ser Thir Luell Arg Asn 13 O 135 14 O

Phe Gly Wall Gly Lys Ser Luell Glu Glin Trp Wall Thir Asp Glu Ala 145 150 155 160

Ser His Luell Asp Ala Lell Thir Ala Glu Ala Gly Arg Pro Luell Asp 1.65 17O 17s US 7,972,785 B2 37 38 - Continued

Pro Tyr Thr Lieu. Lieu. Asn Lys Ala Val Cys Asn Val Ile Ala Ser Lieu. 18O 185 19 O Ile Tyr Ala Arg Arg Phe Asp Tyr Gly Asp Pro Asp Phe Ile Llys Val 195 2OO 2O5 Lieu Lys Ile Leu Lys Glu Ser Met Gly Glu Gln Thr Gly Lieu. Phe Pro 21 O 215 22O Glu Val Lieu. Asn Met Phe Pro Val Lieu. Lieu. Arg Ile Pro Gly Lieu Ala 225 23 O 235 24 O Asp Llys Val Phe Pro Gly Gln Lys Thr Phe Lieu. Thr Met Val Asp Asn 245 250 255 Lieu Val Thr Glu. His Lys Llys Thr Trp Asp Pro Asp Gln Pro Pro Arg 26 O 265 27 O Asp Lieu. Thir Asp Ala Phe Lieu Ala Glu Ile Glu Lys Ala Lys Gly Asn 27s 28O 285 Pro Glu Ser Ser Phe Asn Asp Ala Asn Lieu. Arg Lieu Val Val Asn Asp 29 O 295 3 OO Lieu. Phe Gly Ala Gly Met Val Thir Thr Ser Ile Thr Lieu. Thir Trp Ala 3. OS 310 315 32O Lieu. Lieu. Lieu Met Ile Lieu. His Pro Asp Val Glin Cys Arg Val Glin Glin 3.25 330 335 Glu Ile Asp Glu Val Ile Gly Glin Val Arg His Pro Glu Met Ala Asp 34 O 345 35. O Glin Ala His Met Pro Phe Thr Asn Ala Val Ile His Glu Val Glin Arg 355 360 365 Phe Ala Asp Ile Val Pro Met Asn Lieu Pro His Llys Thir Ser Arg Asp 37 O 375 38O Ile Glu Val Glin Gly Phe Lieu. Ile Pro Lys Gly Thr Thr Lieu. Ile Pro 385 390 395 4 OO Asn Lieu. Ser Ser Val Lieu Lys Asp Glu Thr Val Trp Glu, Llys Pro Lieu 4 OS 41O 415 Arg Phe His Pro Glu. His Phe Lieu. Asp Ala Glin Gly Asn. Phe Wall Lys 42O 425 43 O His Glu Ala Phe Met Pro Phe Ser Ala Gly Arg Arg Ala Cys Lieu. Gly 435 44 O 445 Glu Pro Leu Ala Arg Met Glu Lieu Phe Leu Phe Phe Thr Cys Lieu. Leu 450 45.5 460 Glin Arg Phe Ser Phe Ser Val Pro Thr Gly Glin Pro Arg Pro Ser Asp 465 470 47s 48O Tyr Gly Val Phe Ala Phe Lieu. Leu Ser Pro Ser Pro Tyr Glin Lieu. Cys 485 490 495 Ala Phe Lys Arg SOO

<210s, SEQ ID NO 3 &211s LENGTH: 2505 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 3 Met Glu Glu Val Val Ile Ala Gly Met Ser Gly Lys Lieu Pro Glu Ser 1. 5 1O 15 Glu Asn Lieu. Glin Glu Phe Trp Ala Asn Lieu. Ile Gly Gly Val Asp Met 2O 25 3O Val Thr Asp Asp Asp Arg Arg Trp Lys Ala Gly Lieu. Tyr Gly Lieu Pro 35 4 O 45 US 7,972,785 B2 39 40 - Continued

Arg Ser Gly Lys Lell Lys Asp Luell Ser Lys Phe Asp Ala Ser Phe SO 55 6 O

Phe Gly Wall His Pro Lys Glin Ala His Thir Met Asp Pro Glin Luell Arg 65 70

Lell Luell Luell Glu Wall Ser Glu Ala Ile Wall Asp Gly Gly Ile Asn 85 90 95

Pro Ala Ser Luell Arg Gly Thir Asn Thir Gly Wall Trp Wall Gly Wall Ser 105 11 O

Gly Ser Glu Ala Ser Glu Ala Luell Ser Arg Asp Pro Glu Thir Luell Luell 115 12 O 125

Gly Tyr Ser Met Wall Gly Cys Glin Arg Ala Met Met Ala Asn Arg Luell 13 O 135 14 O

Ser Phe Phe Phe Asp Phe Gly Pro Ser Ile Ala Lell Asp Thir Ala 145 150 155 160

Ser Ser Ser Lell Lell Ala Luell Glin Asn Ala Glin Ala Ile Arg 1.65 17O 17s

Ser Gly Glu Cys Pro Ala Ala Ile Wall Gly Gly Ile Asn Luell Luell Luell 18O 185 19 O

Pro Asn Thir Ser Wall Glin Phe Met Luell Gly Met Luell Ser Pro 195

Asp Gly Thir Ser Phe Asp Asp Ser Gly Asn Gly Arg 21 O 215 22O

Ala Glu Ala Wall Wall Ala Wall Luell Luell Thir Lys Ser Luell Ala Arg 225 23 O 235 24 O

Arg Wall Ala Thir Ile Lell Asn Ala Gly Thir Asn Thir Asp Gly 245 250 255

Glu Glin Gly Wall Thir Phe Pro Ser Gly Glu Ala Glin Glu Glin Luell 26 O 265 27 O

Ile Arg Ser Luell Tyr Glin Pro Gly Gly Wall Ala Pro Glu Ser Luell Glu 27s 285

Ile Glu Ala His Gly Thir Gly Thir Wall Gly Asp Pro Glin Glu 29 O 295 3 OO

Lell Asn Gly Ile Thir Arg Ser Luell Ala Phe Arg Glin Ser Pro Luell 3. OS 310 315

Lell Ile Gly Ser Thir Ser Asn Met Gly His Pro Glu Pro Ala Ser 3.25 330 335

Gly Luell Ala Ala Lell Thir Wall Luell Luell Ser Lell Glu Asn Gly Wall 34 O 345 35. O

Trp Ala Pro Asn Lell His Phe His Asn Pro ASn Pro Glu Ile Pro Ala 355 360 365

Lell Luell Asp Gly Arg Lell Glin Wall Wall Asp Arg Pro Lell Pro Wall Arg 37 O 375

Gly Gly Ile Wall Gly Ile Asn Ser Phe Gly Phe Gly Gly Ala Asn Wall 385 390 395 4 OO

His Wall Ile Luell Glin Pro Asn Thir Glin Glin Ala Pro Ala Pro Ala Pro 4 OS 415

His Ala Ala Luell Pro His Lell Luell His Ala Ser Gly Arg Thir Met Glu 425 43 O

Ala Wall Glin Gly Lell Lell Glu Glin Gly Arg Glin His Ser Glin Asp Luell 435 44 O 445

Ala Phe Wall Ser Met Lell Asn Asp Ile Ala Ala Thir Pro Thir Ala Ala 450 45.5 460

Met Pro Phe Arg Gly Thir Wall Luell Gly Wall Glu Gly His Wall Glin US 7,972,785 B2 41 42 - Continued

465 470

Glu Wall Glin Glin Wall Pro Ala Ser Glin Arg Pro Lell Trp Phe Ile 485 490 495

Ser Gly Met Gly Thir Glin Trp Arg Gly Met Gly Lell Ser Luell Met SOO 505

Lell Asp Ser Phe Glu Ser Ile Luell Arg Ser Asp Glu Ala Luell 515 525

Pro Luell Gly Wall Lys Wall Ser Asp Luell Luell Luell Ser Thir Asp Glu His 53 O 535 54 O

Thir Phe Asp Asp Ile Wall His Ser Phe Wall Ser Lell Thir Ala Ile Glin 5.45 550 555 560

Ile Ala Luell Ile Asp Lell Lell Thir Ser Met Gly Lell Pro Asp Gly 565 st O sts

Ile Ile Gly His Ser Lell Gly Glu Wall Ala Gly Ala Asp Gly 585 59 O

Luell Ser Glin Arg Glu Ala Wall Luell Ala Ala Trp Arg Gly Glin 595 605

Ile Asp Ala Asn Lell Pro Ala Gly Ser Met Ala Ala Wall Gly 610 615

Lell Ser Trp Glu Glu Cys Glin Arg Pro Pro Gly Wall Wall Pro 625 630 635 64 O

Ala His Asn Ser Glu Asp Thir Wall Thir Ile Ser Gly Pro Glin Ala 645 650 655

Ala Wall Asn Glu Phe Wall Glu Glin Luell Glin Glu Gly Wall Phe Ala 660 665 67 O

Glu Wall Arg Thir Gly Gly Luell Ala Phe His Ser Tyr Phe Met Glu 675 685

Gly Ile Ala Pro Thir Lell Lell Glin Ala Luell Lys Wall Ile Arg Glu 69 O. 695 7 OO

Pro Arg Pro Arg Ser Ala Arg Trp Luell Ser Thir Ser Ile Pro Glu Ala 7 Os 71s

Glin Trp Glin Ser Ser Lell Ala Arg Thir Ser Ser Ala Glu Asn Wall 72 73 O 73

Asn Asn Luell Wall Ser Pro Wall Luell Phe Glin Glu Ala Lell Trp His Wall 740 74. 7 O

Pro Glu His Ala Wall Wall Lell Glu Ile Ala Pro His Ala Luell Luell Glin 760 765

Ala Wall Luell Gly Wall Pro Ser Thir Ile Ile Pro Luell 770 775

Met Arg Asp His Lys Asp Asn Luell Glu Phe Phe Lell Thir Asn Luell 79 O 79.

Gly Wall His Lell Thir Gly Ile Asp Ile ASn Pro Asn Ala Luell Phe 805 810 815

Pro Pro Wall Glu Phe Pro Wall Pro Arg Gly Thir Pro Lell Ile Ser Pro 825 83 O

His Ile Lys Trp Asp His Ser Glin Thir Trp Asp Ile Pro Wall Ala Glu 835 84 O 845

Asp Phe Pro Asn Gly Ser Ser Ser Ser Ser Ala Thir Wall Asn Ile 850 855 860

Asp Ala Ser Ser Glu Ser Ser Asp His Tyr Luell Wall Asp His Ile 865 87s

Asp Gly Arg Wall Lell Phe Pro Gly Thir Gly Lell Luell Wall Trp 885 890 895 US 7,972,785 B2 43 44 - Continued Llys Thr Lieu Ala Arg Ser Lieu. Ser Lieu. Ser Lieu. Glu Glu Thr Pro Val 9 OO 905 91 O Val Phe Glu Asn Val Thr Phe His Glin Ala Thir Ile Leu Pro Arg Thr 915 92 O 925 Gly Thr Val Pro Lieu. Glu Val Arg Lieu. Lieu. Glu Ala Ser His Ala Phe 93 O 935 94 O Glu Val Ser Asp Ser Gly Asn Lieu. Ile Val Ser Gly Llys Val Tyr Glin 945 950 955 96.O Trp Glu Asp Pro Asp Ser Llys Lieu. Phe Asp His Pro Glu Val Pro Ile 965 97O 97. Pro Ala Glu Ser Glu Ser Val Ser Arg Lieu. Thr Glin Gly Glu Val Tyr 98O 985 99 O Lys Glu Lieu. Arg Lieu. Arg Gly Tyr Asp Tyr Gly Pro His Phe Glin Gly 995 1OOO 1005 Val Tyr Glu Ala Thir Lieu. Glu Gly Glu Glin Gly Llys Lieu Lleu Trp

Lys Asp Asn Trp Val Thr Phe Met Asp Thr Met Leu Glin Ile Ser O25 O3 O O35 Ile Leu Gly Phe Ser Lys Glin Ser Leu Gln Leu Pro Thr Arg Val O4 O O45 OSO Thr Ala Ile Tyr Ile Asp Pro Ala Thr His Leu Gln Llys Val Tyr O55 O6 O O65 Met Lieu. Glu Gly Asp Thr Glin Val Ala Asp Val Thir Thr Ser Arg Of O O7 O8O Cys Lieu. Gly Val Thr Val Ser Gly Gly Val Tyr e Ser Arg Lieu. O85 O9 O O95 Gln Thr Thr Ala Thr Ser Arg Arg Glin Glin Glu Gln Leu Val Pro OO O5 10 Thr Lieu. Glu Lys Phe Val Phe Thr Pro His Val Glu Pro Glu. Cys 15 2O 25 Lieu. Ser Glu Ser Ala Ile Lieu Gln Lys Glu Lieu Gln Lieu. Cys Llys 3O 35 4 O Gly Lieu Ala Lys Ala Lieu. Glin Thir Lys Ala Thr Glin Glin Gly Lieu.

Llys Met Thr Val Pro Gly Lieu. Glu Asp Lieu Pro Glin His Gly Lieu. 60 65 70 Pro Arg Lieu. Lieu Ala Ala Ala Cys Glin Lieu. Glin Lieu. Asn Gly Asn

Lieu. Glin Lieu. Glu Lieu. Gly Glu Val Lieu Ala Arg Glu Arg Lieu. Lieu. 90 95 2OO Lieu Pro Glu Asp Pro Lieu e Ser Gly Lieu. Lieu. Asn. Ser Glin Ala 2O5 21 O 215 Lieu Lys Ala Cys Ile Asp Thir Ala Lieu. Glu Asn Lieu. Ser Thr Lieu. 22O 225 23 O Llys Met Llys Val Val Glu Val Lieu Ala Gly Glu Gly His Lieu. Tyr 235 24 O 245

Ser His Ile Ser Ala Lieu. Lieu. Asn Thr Glin Pro Met Lieu. Glin Lieu. 250 255 26 O Glu Tyr Thr Ala Thr Asp Arg His Pro Glin Ala Lieu Lys Asp Val 265 27 O 27s Glin Thir Lys Lieu. Glin Gln His Asp Val Ala Glin Gly Glin Trp Asp 28O 285 29 O

Pro Ser Gly Pro Ala Pro Thr Asn Lieu. Gly Ala Lieu. Asp Lieu Val 295 3OO 305 US 7,972,785 B2 45 46 - Continued

Wall Cys Asn Ala Luell Ala Thir Luell Gly Asp Pro Ala Lell Ala 310 315 32O

Lell Asn Met Wall Ala Luell Asp Gly Phe Lell Lell 335

Met Thir Wall Luell Lys His Ala Luell Gly Glu Thir Lell Ala 350

Luell Pro Ser Glu Wall Glin Pro Gly Pro Ser Phe Luell Ser Glin 355 360 365

Glu Glu Trp Glu Ser Luell Phe Ser Arg Ala Lell His Lell Wall 37O 375 38O

Gly Luell Ser Phe yr Gly Thir Ala Lell Phe Luell Arg 385 390 395

Arg Luell Ser Pro Glin Asp Pro Ile Phe Lell Pro Wall Glu Asp 4 OO 41 O

Thir Ser Phe Glin Trp Wall Ser Luell Ser le Luell Ala Thir 415 425

Ser Ser Ser Glin Pro Wall Luell Thir Ala Met Asn Pro Thir 43 O 44 O

Ser Wall Wall Gly Luell Asn Luell Arg Glu Pro Gly 45.5

Gly Arg Ile Arg Luell Luell Ser Asn Lell Ser Ser Thir 47 O

Ser Wall Pro Luell Pro Gly Ser Ser Glu Luell Glin 485

Wall Glu Ser Asp Luell Met Asn Wall Asp Gly Ala

Trp Ala Phe Arg His Phe Glin Luell Glu Glin Pro Glu 510

Glu Thir Ala His Ala Phe Wall Asn Wall Lell Arg Gly Asp 525

Lell Ser Ile Arg Trp Ser Ser Pro Lell His Met Glin

Pro Pro Ser Ser Ser Gly Glin Luell Thir Ala 550

Ser Luell Asn Phe Arg Asp Met Luell Ala Thir Lell Ser 565

Pro Ala Ile Pro Gly Trp Ala Ser Arg Met Lell

Gly Met Glu Phe Ser Gly Asp Gly Arg Wall Met 595

Gly Wall Pro Ala Glu Luell Ala Thir Ser Wall Luell Lell Ser 62O

Pro Phe Lell Trp Asp Pro Ser Ser Trp Luell Glu Glu 635

Ala Ser Wall Pro Wall Tyr Thir Thir Ala yr Ser Lell 650

Wall Arg Gly Arg Ile His Gly Glu Thir Wall Luell Ile His 665

Ser Ser Gly Gly Wall Glin Ala Ala Ile Ile Ala Lell

Ser Luell Gly Arg Wall Phe Thir Thir Wall Gly Ala Glu 685 69 O.

Arg Ala Lell Glin Ala Arg Phe Pro Glin Lell Asp Thir Ser US 7,972,785 B2 47 48 - Continued

7 OO 7Os

Phe Asn Ser Arg Asp Ser Phe Glu Glin His Wall Lell Lell 72 O 72

His Gly Gly Gly Asp Luell Wall Lell Asn Ser Lell Ala 73 O 73 74 O

Glu Glu Lell Glin Ala Ser Wall Arg Lell Ala Glin His Gly 74. 7 O 7ss

Arg Phe Lell Glu Ile Gly Phe Asp Luell Ser Asn ASn His Pro 760 765 770

Lell Met Ala Ile Phe Luell Asn Wall Thir Phe His Gly Ile 775 78O 78s

Lell Luell Asp Ala Luell Phe Glu Gly Ala ASn Asp Trp Arg Glu 79 O 79.

Wall Glu Lell Luell Lys Gly Ile Arg Asp Wall Wall 810

Pro Thir Wall Phe Pro Ala Glin Glu Asp Ala 825

Phe Met Ala Glin Lys His Ile Gly Wall Lell Wall

Glin Arg Glu Glu Glu Glu Ala Met Lell Gly Ala Glin

Pro Lell Ile Ser Ala Ser Thir Phe Pro Glu His 865

Ser Ile Ile Thir Gly Luell Gly Gly Gly Lell Glu

Lell Arg Trp Luell Wall Arg Gly Ala Glin Luell Wall Lell 895

Thir Ser Arg Ser Gly Ile Thir Gly Glin Ala His Wall 910 92 O

Arg Glu Trp Arg Arg Glin Ile His Wall Lell Wall Ser Thir Ser 925 935

Asn Ser Ser Luell Glu Ala Arg Ala Lell le Ala Glu Ala 950

Thir Lell Gly Pro Wall Gly Wall Phe Asn Lell Ala Met Wall 965

Lell Asp Ala Met Luell Glu Asn Glin Thir Pro Glu Luell Phe Glin 97. 98 O

Asp Asn Pro Lys yr Asn Gly Thir Lell Asn Luell Asp Arg 990 995

Ala Arg Glu Ala Cys Pro Glu Luell Asp Phe Wall Ala Phe 2005 2010

Ser Ser Wall Ser Gly Gly Asn Ala Glin Ser Asn 2015 2O25

Gly Phe Ala Asn Ser Thir Met Glu Arg Ile Glu Glin Arg Arg 2O3O 2O35 2O4. O

His Asp Gly Lell Pro Gly Luell Ala Wall Glin Trp Gly Ala Ile Gly 2O45 2OSO 2O55

Asp Wall Gly Ile Ile Luell Ala Met Gly Thir Asn Asp Thir Wall 2O60 2. Of O

Wall Gly Thir Luell Pro Glin Arg Ile Ser Ser Met Glu Wall 2O8 O

Lell Asp Lell Phe Luell Asn Glin Pro His Ala Wall Lell Ser Ser Phe 2O90 2095 21OO

Wall Luell Wall Glu Lys Ala Wall Ala His Gly Asp Gly Glu Ala 2105 211 O 2115 US 7,972,785 B2 49 50 - Continued

Glin Asp Lell Wall Lys Ala Wall Ala His Ile Lell Gly Ile Arg 2125 213 O

Asp Ala Gly Ile Asn Luell Asp Ser Ser Lell Ala Asp Lell Gly 214 O 2145

Lell Ser Lell Met Gly Wall Glu Wall Arg Glin Ile Luell Glu Arg 215.5 216 O

Glu Asp Lell Wall Luell Pro Ile Arg Glu Wall Arg Glin Lell Thir 217 O 21.75

Lell Lell Glin Glu Met Ser Ser Ala Gly Ser Asp Thir 21.85 219 O

Glu Ala Ala Ser Asn Asp Thir Ser Luell Glin 22 OO 22O5

Ala Lell Asn Lel Ser Ile Luell Luell Wall Asn Pro Glu Gly Pro 2215 222 O

Thir Luell Thir Arg Lel Asn Ser Wall Glin Ser Ser Glu Arg Pro Lell 2225 223 O 2235

Phe Luell Wall His Ile Glu Gly Ser Ile Thir Wall Phe His Ser 224 O 2.245 225 O

Lell Ala Ala Lel Ser Wall Pro Thir Gly Lell Glin Thir 2255 226 O 2265

Glin Ala Ala Pro Lel Asp Ser Ile Pro ASn Lell Ala Ala 2270 2275 228O

Ile Asp Ile Glin Wall Glin Pro Glu Gly Pro His Wall 2285 229 O 2295

Ala Gly Ser Phe Gly Ala Cys Wall Ala Phe Glu Met Ser 23 OO 23 OS 2310

Glin Luell Glin Ala Glin Glin Gly Pro Ala Pro Ala His ASn Asn Lell 2315 232O 2325

Phe Luell Phe Asp Gly Ser His Thir Wall Lell Ala Thir Glin 233 O 2335 234 O

Ser Tyr Arg Ala Luell Thir Pro Cys Glu Ala Glu Ala Glu 2345 2350 2355

Ala Glu Ala Ile Phe Phe Ile Glin Phe Wall Asp Ala Glu 2360 23.65 2370

His Ser Wall Luell Glu Ala Luell Luell Pro Lell Lys Ser Lell Glu 2375 238O 23.85

Asp Arg Wall Ala Ala Ala Wall Asp Luell Ile Thir Arg Ser His Glin 23.90 23.95 24 OO

Ser Luell Asp Arg Arg Asp Luell Ser Phe Ala Ala Wall Ser Phe 24 O5 241. O 24.15

Lys Lell Arg Ala Ala Asp Glin Tyr Pro Lys Ala 242O 24.25 243 O

His Gly Asn Wall Ile Luell Luell Arg Ala Thir Gly Gly Thir 2435 244 O 2445

Gly Glu Asp Lell Gly Ala Asp Tyr Asn Luell Ser Glin Wall Asp 2450 2455 246 O

Gly Lys Wall Ser Wall His Ile Ile Glu Gly Asp His Arg Thir Lell 24 65 2470 2475

Lell Glu Gly Arg Gly Luell Glu Ser Ile Ile Asn Ile Ile His Ser 248O 2485 249 O

Ser Luell Ala Glu Pro Arg Wall Ser Wall Arg Glu Gly 2495 25 OO 2505 US 7,972,785 B2 51 52 - Continued <210s, SEQ ID NO 4 &211s LENGTH: 381 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 4.

Met Asn. Pro Phe Ser Ser Glu Ser Ala Trp Luell Lell Thir Ala Thir 1. 5 15

Ala Wall Luell Gly Gly Met Lell Luell Cys Lys Ala Trp Ser Ser Gly Glin 2O 25

Lell Arg Ser Glin Wall Wall Luell Ala Gly Luell Trp Gly Gly Ala 35 4 O 45

Lell Luell Ser Luell Ser Lell Lell Ser Luell Phe Lell Lell Ser Wall Ser SO 55 6 O

Cys Phe Phe Luell Lell Tyr Wall Ser Ser Ser Asp Glin Asp Luell Luell Pro 65 70

Wall Asp Glin Ala Wall Lell Wall Thir Gly Ala Asp Ser Gly Phe Gly 85 90 95

His Ala Luell Ala Lys His Lell Asp Lys Luell Gly Phe Thir Wall Phe Ala 105 11 O

Gly Wall Luell Asp Glu Gly Pro Gly Ala Glu Glu Lell Arg Asn 115 12 O 125

Ser Glu Arg Lell Ser Wall Luell Glin Met Asp Wall Thir Pro Glu 13 O 135 14 O

Glin Ile Asp Wall His Ser Glu Wall Ala Glu Ile Glin Asp Lys 145 150 155 160

Gly Luell Trp Ala Wall Wall Asn Asn Ala Gly Wall Lell His Phe Pro Ile 1.65 17O 17s

Asp Gly Glu Luell Ile Pro Met Thir Wall Tyr Arg Met Ala Wall 18O 185 19 O

Asn Phe Phe Gly Ala Wall Glu Wall Thir Wall Phe Lell Pro Luell Luell 195 2O5

Arg Lys Ser Gly Arg Lell Wall Asn Wall Ser Ser Met Gly Ala Met 21 O 215 22O

Ile Pro Phe Glin Met Wall Ala Ala Ala Ser Thir Ala Ala Ile 225 23 O 235 24 O

Ser Met Phe Ser Ala Wall Ile Arg Glu Luell Ala Trp Gly Wall 245 250 255

Wall Wall Thir Ile His Pro Gly Phe Glin Thir Asn Ile Wall Gly 26 O 27 O

Ser Glin Asp Ser Trp Asp Met Glu Ile Lell Asp His Phe 285

Ser Lys Glu Ile Glin Glu Asn Tyr Glin Glu Tyr Wall His Thir Glin 29 O 295 3 OO

Lys Luell Ala Luell Pro Wall Met Arg Met Ser Asn Pro Asp Ile Thir 3. OS 310 315

Pro Wall Luell Arg Asp Ile Glin His Ile Ala Asn Pro Ser 3.25 330 335

Ser Phe Cys Ser Gly Arg Met Thir Tyr Luell Trp Ile Cys Phe Ala 34 O 345 35. O

Ala Ser Pro Ile Ser Lell Luell Asp Tyr Ile Lell Lys Asn Phe 355 360 365

Thir Pro Luell Met Pro Arg Ala Luell Arg Thir Ala Ser 37 O 375 38O US 7,972,785 B2 53 54 - Continued <210s, SEQ ID NO 5 &211s LENGTH: 295 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 5 Met Glu. Thir Ser Met Pro Glu Tyr Tyr Glu Wall Phe Gly Asp Phe His 1. 5 1O 15

Gly Phe Lieu Met Asp Llys Lieu. Phe Thr Lys Tyr Trp Glu Asp Wall Glu 2O 25 3O

Thr Phe Ser Ala Arg Pro Asp Asp Lieu. Lieu. Wall Wall Thir Tyr Pro 35 4 O 45

Ser Gly Ser Thr Trp Ile Gly Glu Ilie Wall Asp Met Ile Glu SO 55 6 O

Gly Asp Val Glu Lys Cys Llys Glu Asp Ala Ile Phe Asn Arg Ile Pro 65 70 8O

Tyr Lieu. Glu. Cys Arg Asn. Glu Asp Lieu. Ile ASn Gly Ile Lys Glin Luell 90 95

Lys Glu Lys Glu Ser Pro Arg Ile Val Lys Thir His Lell Pro Ala 1OO 105 11 O

Lieu. Leu Pro Ala Ser Phe Trp Glu Lys Asn Cys Ile Ile Tyr Luell 115 12 O 125

Cys Arg Asn Ala Lys Asp Wall Wall Wal Ser Tyr Tyr Tyr Phe Phe Luell 13 O 135 14 O

Ile Met Lys Ser Tyr Pro Asn Pro Llys Ser Phe Ser Glu Phe Wall Glu 145 150 155 160

Llys Phe Met Glu Gly Glin Val Pro Tyr Gly Ser Trp Tyr Asp His Wall 1.65 17O 17s

Llys Ser Trp Trp Glu Lys Ser Lys Asn. Ser Arg Wall Lell Phe Met Phe 18O 185 19 O

Tyr Glu Asp Met Lys Glu Asp Ile Arg Arg Glu Wall Wall Llys Lieu. Ile 195 2OO

Glu Phe Lieu. Glu Arg Asp Pro Ser Ala Glu Luell Wall Asp Arg Ile Ile 21 O 215 22O

Gln His Thir Ser Phe Glin Glu Met Lys Asn ASn Pro Thir Asn Tyr 225 23 O 235 24 O

Ser Met Lieu Pro Glu. Thir Met Ile Asp Lieu. Lys Wall Ser Pro Phe Met 245 250 255

Arg Lys Gly Ile Val Gly Asp Trp Lys Asn His Phe Pro Glu Ala Luell 26 O 265 27 O

Arg Glu Arg Phe Glu Glu. His Tyr Glin Glin Glin Met Lys Asp Cys Pro 27s 28O 285 Val Llys Phe Arg Ala Glu Lieu 29 O 295

<210s, SEQ ID NO 6 &211s LENGTH: 1585 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 6

Met Ser Ser Lieu. Glin Glu Lieu. Cys Ser Gly Lieu Pro Lell Arg Pro Luell 1. 5 15

Pro Glu Asn Arg Gly Arg Trp Ala Gly Val Pro His Ala Pro Wall Arg 2O 25 3O

Thr Pro Asp Leu Ser Pro Glu Glu Glu Glin Lieu. Ala Lell Arg Asn Ala 35 4 O 45 US 7,972,785 B2 55 56 - Continued

Lell Arg Phe Pro Pro Asp Wall Glin Luell Lell Ala Luell Glu Phe SO 55 6 O

Ala Glin Glu Luell Arg Glin Phe Gly His Ile Tyr Met Arg Phe Cys 65 70

Pro Ser Ile Glu Met Arg Ala Pro Ile Glu Glin Pro Cys Arg 85 90 95

Thir Arg Ala Ala Ala Ala Ile Met His Met Ile Met Asn Asn Luell Asp 105 11 O

Pro Ala Wall Ala Glin Phe Pro Glin Glu Luell Wall Thir Tyr Gly Gly Asn 115 12 O 125

Gly Glin Wall Phe Ser Asn Trp Ala Glin Wall Trp Wall Glin Gly Ala Ser 13 O 135 14 O

Ser His Luell Glin Ser Pro Pro Ala Luell Ser Ile Glin Phe Trp Luell 145 150 155 160

Thir Met Ser Lell Ser Met Thir Glu Glu Glin Thir Luell Wall Met 1.65 17O 17s

Ser Gly His Pro Lell Gly Luell Phe Pro Ser Ser Ser His Ala Pro 18O 185 19 O

Arg Luell Wall Ile Thir Asn Gly Met Wall Ile Pro Asn Tyr Ser Ser Arg 195

Thir Glu Tyr Glu Lell Phe Ala Luell Gly Wall Thir Met Gly Glin 21 O 215

Met Thir Ala Gly Ser Tyr Ile Gly Pro Glin Gly Ile Wall His 225 23 O 235 24 O

Gly Thir Wall Luell Thir Wall Lell Asn Ala Gly Arg Arg Luell Gly Ile 245 250 255

Glu Asp Luell Ala Gly Wall Phe Wall Thir Ser Gly Lell Gly Gly Met 26 O 265 27 O

Ser Gly Ala Glin Ala Ala Ala Wall Ile Wall Gly Cys Ile Gly Wall 285

Ile Ala Glu Wall Asp Ala Ala Luell Wall Arg His Glin Glin Gly 29 O 295 3 OO

Trp Luell Met Glu Wall Thir Asp Ser Luell Asp His Ile Ala Arg Luell 3. OS 310 315

Arg Glu Ala Arg Lys Glu Wall Luell Ser Lell Gly His Gly 3.25 330 335

Asn Wall Wall Asp Lell Trp Glu Arg Luell Wall His Glu Lell Asp Thir Thir 34 O 345 35. O

Gly Glu Luell Luell Wall Asp Lell Gly Ser Asp Glin Thir Ser His Asn 355 360 365

Pro Phe Asn Gly Gly Tyr Tyr Pro Wall Glin Luell Ser Phe Ser Glu Ala 37 O 375

Glin Ser Luell Met Ser Ser Asn Pro Ala Ala Phe Asn Luell Wall Glin 385 390 395 4 OO

Glu Ser Luell Arg Arg His Ile Ser Ala Ile ASn Arg Lell Ala Glin Glu 4 OS 415

Phe Phe Phe Trp Asp Gly Asn Ala Phe Lell Lell Glu Ala Glin 425 43 O

Arg Ala Gly Ala Asp Wall Glu Lys Gly Ala Asn Lys Thir Glu Phe 435 44 O 445

Arg Tyr Pro Ser Tyr Wall Glin His Ile Met Gly Asp Ile Phe Ser Glin 450 45.5 460

Gly Phe Gly Pro Phe Arg Trp Wall Thir Ser Gly Asn Pro Glin Asp US 7,972,785 B2 57 58 - Continued

465 470

Lell Thir Wall Thir Asp His Lell Ala Thir Ser Wall Lell Glu Ala Ile 485 490 495

Ala Asp Gly Gly Asp Thir Ser Gly Trp Wall Wall Lell Arg Ser Asp Ser SOO 505

Phe Cys Gly Ala Lell Gly Gly Luell Glin Ile Lell Glin Glu Ser 515 525

Asp His Arg Lell Lell His Gly Thir Ser Ser Thir Phe Arg Ser 53 O 535 54 O

His Ser Luell Ala Wall Lell Met Ala Ser Wall Luell Glin Tyr 5.45 550 555 560

Met Asp Asn Ile Arg Trp Ile Arg Glu Ala Ala His Glin Luell Wall 565 st O sts

Wall Gly Ser Glin Ala Arg Ile Luell Tyr Ser Asp Glin Gly Arg Wall 585 59 O

Ala Ile Ala Wall Ala Ile Asn Glin Ala Ile Ala Ser Gly Ile 595 605

Ala Pro Wall Wall Lell Ser Arg Asp His His Asp Wall Ser Gly Thir Asp 610 615

Ser Pro Phe Arg Glu Thir Ser Asn Ile Tyr Asp Gly Ser Ala Phe Cys 625 630 635 64 O

Ala Asp Met Ala Wall Glin Asn Phe Wall Gly Asp Ala Arg Gly Ala 645 650 655

Thir Trp Wall Ala Lell His Asn Gly Gly Gly Wall Gly Trp Gly Glu Wall 660 665 67 O

Ile Asn Gly Gly Phe Gly Lell Wall Luell Asp Gly Thir Pro Glu Ala Glu 675 685

Glin Lys Ala Arg Met Met Lell Ser Trp Asp Wall Ser Asn Gly Wall Ala 69 O. 695 7 OO

Arg Arg Trp Ser Gly Asn Pro Ala Tyr Glu Ile Ile Glin 7 Os

Thir Met His Asp Asn Ser Gly Luell Wall Wall Thir Lell Pro His Glu Wall 72 73 O 73

Ala Asp Glu Glin Wall Lell Arg Glin Asp Luell Luell Glin Gly Lys Pro Gly 740 74. 7 O

Lell Arg Ala Luell Glin Arg Gly His Arg Asp Glu Lys Glin Ser Arg 760 765

Glu Wall Glin Ser Lell Ser Ala Ser Ser Pro Cys Asp Arg Arg Luell 770 775

Gly Luell Ala His Ser Glin Luell Gly Pro Ala Pro Ala Pro 79 O 79.

Pro Pro Gly Ser Lell Wall Thir Thir Wall Thir Ala Lell Ala Arg His 805 815

Ala His Ala His Gly Pro Arg Ala Arg Ala Arg Asp Ala Thir Luell 825 83 O

Gly Thir Luell Arg Lell Ala Luell Thir Thir Pro Gly Gly Arg Thir 835 84 O 845

Lell Gly Wall Ala Arg Met Pro Gly Arg Gly Pro Lell Phe Ser Gly 850 855 860

Gly Cys Gly Ser Asp Ala Ser Ala Gly Pro Lys Met Asp Luell Pro Ala 865

Wall Luell Ala Ala Pro Ala Thir Arg Gly Asp Glin His Gly Gly Gly Pro 885 890 895 US 7,972,785 B2 59 - Continued Ser Arg Lieu. Arg Arg Gly Thr Gly Pro Ser Lieu. Gly Ala Gly Pro Gly 9 OO 905 91 O Arg Arg Arg Lieu Lleu Lleu Lieu. Arg Ser Pro Glu Asp Gly Gly Pro Gly 915 92 O 925 Pro Arg Glin Glu Glu Ala Pro Gly Pro Ser Pro Pro Pro Pro Glu Asp 93 O 935 94 O Gly Gly Asp Ser Phe Val Val Lieu. Lieu. Glu Val Pro Arg Ala Ala Asp 945 950 955 96.O Thr His Glu Gln Glu Glu Thr Glu Pro Asp Ser Gly Ala Ser Pro Thr 965 97O 97. Glu Glin Val Pro Ala Ala Ala Pro Gly Ala Ala Lieu Ala Gly Thr Val 98O 985 99 O Thir Ile His Asn. Glin Asp Lieu. Lieu Val Arg Phe Asp Arg Gly Val Phe 995 1OOO 1005 Thir Lieu Ala Ala Ala Pro Ala Pro Ala Thr Glin Gly Lieu. His Pro O1O O15 O2O Ala Thir Thr Pro Gly Lieu. Glu Pro Ser Ser Ala Ala Ala Ser Arg O25 O3 O O35 Arg Gly Pro Val Ala Ala Ser Ala Gly Ser Pro Ala Tyr Arg Cys O4 O O45 OSO Pro Glu Pro Glin Cys Ala Lieu. Ser Phe Ala Lys Llys His Glin Lieu. O55 O6 O O65 Llys Val His Lieu. Lieu. Thir His Gly Ser Lieu. Glin Gly Arg Arg Pro

Phe Lys Cys Pro Leu Asp Gly Cys Gly Trp Ala Phe Thr Thr Ser

Tyr Lys Lieu Lys Arg His Lieu. Glin Ser His Asp Llys Lieu. Arg Pro

Phe Ser Cys Pro Val Gly Gly Cys Gly Lys Llys Phe Thr Thr Val

Tyr Asn Lieu Lys Ala His Met Lys Gly His Glu Glin Glu Ser Lieu.

Phe Lys Cys Glu Val Cys Ala Glu Arg Phe Pro Thr His Ala Lys

Lieu. Asn Ser His Glin Arg Ser His Phe Glu Pro Glu Arg Pro Tyr

Lys Cys Asp Phe Pro Gly Cys Glu Lys Thr Phe e Thir Wal Ser

Ala Lieu. Phe Ser His Asn Arg Ala His Phe Arg Glu Glin Glu Lieu.

Phe Ser Cys Ser Phe Pro Gly Cys Asn Lys Glin Tyr Asp Lys Ala 2O5 21 O 215 Cys Arg Lieu Lys Ile His Lieu. Arg Ser His Thr Gly Glu Arg Pro 22O 225 23 O Phe Ile Cys Asp Ser Asp Ser Cys Gly Trp Thr Phe Thr Ser Met 235 24 O 245 Ser Lys Lieu. Lieu. Arg His Lys Arg Llys His Asp Asp Asp Arg Arg

Phe Thr Cys Pro Val Glu Gly Cys Gly Lys Ser Phe Thr Arg Ala 265 27 O 27s Glu. His Leu Lys Gly His Ser Ile Thr His Leu Gly Thr Lys Pro 28O 285 29 O Phe Glu. Cys Pro Val Glu Gly Cys Cys Ala Arg Phe Ser Ala Arg 295 3OO 305 US 7,972,785 B2 61 - Continued

Ser Ser Lieu. Tyr Ile His Ser Lys Llys His Lieu. Glin Asp Val Gly 310 315 32O Thr Pro Llys Ser Arg Cys Pro Val Ser Ser Cys Asn Arg Lieu Phe 3.25 33 O 335 Thir Ser Lys His Ser Met Lys Ala His Met Val Arg Gln His Ser 34 O 345 350 Arg Arg Glin Asp Lieu Val Pro Glin Lieu. Glu Ala Pro Ser Ser Lieu. 355 360 365 Thr Pro Ser Ser Glu Lieu Ser Ser Pro Gly Glin Ser Glu Lieu. Thr 37O 375 38O Asn. Ile Asp Lieu Ala Ala Lieu. Phe Ser Asp Thr Pro Ala Asn. Ser 385 390 395 Ser Ser Ser Thr Ala Gly Ser Asp Glu Ala Lieu. Asn. Ser Gly Ile 4 OO 405 41 O Lieu. Thir Ile Asp Val Thir Ser Val Ser Ser Ser Lieu. Gly Gly Asn 415 42O 425 Lieu Pro Thr Asn Asn Asn Ser Leu Gly Pro Met Asp Pro Leu Val 43 O 435 44 O Lieu Val Ala His Ser Asp Met Pro Pro Ser Lieu. Asp Ser Pro Lieu. 445 450 45.5 Val Lieu. Gly Thr Pro Ala Thr Val Lieu. Glin Pro Gly Ser Phe Ser 460 465 47 O Ala Asp Asp Ser Glin Ala Met Ser Thr Gly Ala Val Gly Cys Lieu. 47s 48O 485 Val Ala Lieu Pro Met Arg Asn Lieu. Ser Glin Asp Ser Pro Ala Lieu. 490 495 SOO Thr Pro Ser Asn Asn Lieu. Thir Ala Leu Gly Thr Thr Pro Thir Ser 5 OS 510 515 Ser Asp Thr Thr Glin Glu Thir Ala Ser Val Pro Asp Leu Lleu Val 52O 525 53 O Pro Ile Llys Val Glu Glin Asp Lieu. Ser Pro Val Pro Asp Val Val 535 54 O 545 Gln Gly Gln Lys Glu Ser His Gly Pro Ser Val Lieu Ser Ser Ser 550 555 560 Ala Glu Arg Lieu. Gly Ala Glin Lys Asp Ser Glu Lieu. Ser Ala Gly 565 st O sts Thr Gly Ser Lieu. Tyr Lieu Val

<210s, SEQ ID NO 7 &211s LENGTH: 113 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OO > SEQUENCE: 7 Met Ala Ala Lys Thr Gly Ser Gln Leu Glu Arg Ser Ile Ser Thr Ile 1. 5 1O 15 Ile Asin Val Phe His Glin Tyr Ser Arg Llys Tyr Gly His Pro Asp Thr 2O 25 3O Lieu. Asn Lys Ala Glu Phe Lys Glu Met Val Asn Lys Asp Lieu Pro Asn 35 4 O 45 Phe Lieu Lys Arg Glu Lys Arg Asn. Glu Asn Lieu. Lieu. Arg Asp Ile Met SO 55 6 O

Glu Asp Lieu. Asp Thr Asn Glin Asp Asn Glin Lieu. Ser Phe Glu Glu. Cys 65 70 7s 8O US 7,972,785 B2 63 64 - Continued

Met Met Lieu Met Gly Llys Lieu. Ile Phe Ala Cys His Glu Lys Lieu. His 85 90 95 Glu Asn. Asn Pro Arg Gly. His Asp His Arg His Gly Lys Gly Cys Gly 105 11 O Lys

SEQ ID NO 8 LENGTH: TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 8

Met Glu Glu Glu Ile Ala Ala Luell Wall Ile Asp Asn Gly Ser Gly Met 1. 5 15

Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Wall Phe Pro 2O 25

Ser Ile Wall Gly Arg Pro Arg His Glin Gly Wall Met Wall Gly Met Gly 35 4 O 45

Glin Lys Asp Ser Tyr Wall Gly Asp Glu Ala Glin Ser Arg Gly Ile SO 55 6 O

Lell Thir Luell Tyr Pro Ile Glu His Gly Ile Wall Thir Asn Trp Asp 65 70

Asp Met Glu Ile Trp His His Thir Phe Asn Glu Luell Arg Wall 85 90 95

Ala Pro Glu Glu His Pro Wall Luell Luell Thir Glu Ala Pro Luell Asn Pro 105 11 O

Ala Asn Arg Glu Met Thir Glin Ile Met Phe Glu Thir Phe Asn 115 12 O 125

Thir Pro Ala Met Tyr Wall Ala Ile Glin Ala Wall Lell Ser Luell Ala 13 O 135 14 O

Ser Gly Arg Thir Thir Gly Ile Wall Met Asp Ser Gly Asp Gly Wall Thir 145 150 155 160

His Thir Wall Pro Ile Glu Gly Ala Luell Pro His Ala Ile Luell 1.65 17O 17s

Arg Luell Asp Luell Ala Arg Asp Luell Thir Asp Lell Met Ile 18O 185 19 O

Lell Thir Glu Arg Gly Ser Phe Thir Thir Thir Ala Glu Arg Glu Ile 195

Wall Arg Asp Ile Lys Glu Lys Luell Wall Ala Lell Asp Phe Glu 21 O 215

Glin Glu Met Ala Thir Ala Ala Ser Ser Ser Ser Lell Glu Ser Tyr 225 23 O 235 24 O

Glu Luell Pro Asp Gly Glin Wall Ile Thir Ile Gly Asn Glu Arg Phe Arg 245 250 255

Pro Glu Ala Lell Phe Glin Pro Ser Phe Luell Gly Met Glu Ser 26 O 265 27 O

Gly Ile His Glu Thir Thir Phe Asn Ser Ile Met Cys Asp Wall Asp 27s 285

Ile Arg Asp Lell Ala Asn Thir Wall Luell Ser Gly Gly Thir Thir 29 O 295 3 OO

Met Pro Gly Ile Ala Asp Arg Met Glin Lys Glu Ile Thir Ala Luell 3. OS 310 315 32O

Ala Pro Ser Thir Met Ile Lys Ile Ile Ala Pro Pro Glu Arg 3.25 330 335 US 7,972,785 B2 65 66 - Continued Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe 34 O 345 35. O Gln Glin Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser 355 360 365

Ile Wall His Arg Llys Cys Phe 37 O 375

SEO ID NO 9 LENGTH: TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 9

Met Ser Ser Pro Ala Glin Pro Ala Wall Pro Ala Pro Lell Ala Asn Luell 1. 15

Lys Ile Glin His Thir Ile Phe Ile Asn ASn Glu Trp His Asp Ser 25 3O

Wall Ser Gly Lys Phe Pro Wall Luell Asn Pro Ala Thir Glu Glu Wall 35 4 O 45

Ile Cys His Wall Glu Glu Gly Asp Ala Asp Wall Asp Ala Wall SO 55 6 O

Lys Ala Ala Arg Glin Ala Phe Glin Ile Gly Ser Pro Trp Arg Thir Met 65 70

Asp Ala Ser Glu Arg Gly Arg Luell Luell Asn Lys Lell Ala Asp Luell Met 85 90 95

Glu Arg Asp Arg Lell Lell Lell Ala Thir Ile Glu Ala Ile Asn Gly Gly 105 11 O

Wall Phe Ala Asn Ala Luell Ser Asp Luell Gly Gly Ser Ile 115 12 O 125

Ala Luell Cys Ala Gly Trp Ala Asp Ile His Gly Glin Thir 13 O 135 14 O

Ile Pro Ser Asp Asp Ile Phe Thir Phe Thir Arg Arg Glu Pro Ile 145 150 155 160

Gly Wall Gly Ile Ile Pro Trp Asn Phe Pro Lell Luell Met Phe 17O

Ile Trp Ile Pro Ala Luell Ser Gly Asn Thir Wall Wall Wall 18O 185 19 O

Pro Ala Glu Thir Pro Luell Thir Ala Luell His Met Ala Ser Luell 195

Ile Lys Glu Ala Phe Pro Pro Gly Wall Wall Asn Ile Wall Pro Gly 21 O 215 22O

Tyr Gly Pro Thir Gly Ala Ala Ile Ser Ser His Met Asp Wall Asp 225 23 O 235 24 O

Wall Ala Phe Thir Gly Ser Thir Glin Wall Gly Lell Ile Lys Glu 245 250 255

Ala Ala Gly Lys Ser Asn Lell Arg Wall Thir Lell Glu Luell Gly Gly 26 O 265 27 O

Ser Pro Ile Wall Phe Ala Asp Ala Asp Lell Asp Ile Ala Wall 27s 28O 285

Glu Phe Ala His His Gly Wall Phe Tyr His Glin Gly Glin Wall 29 O 295 3 OO

Ala Ala Ser Arg Ile Phe Wall Glu Glu Ser Wall Asp Glu Phe Wall 3. OS 310 315

Arg Ser Wall Glu Arg Ala Tyr Wall Lell Gly Asn Pro Luell 3.25 330 335 US 7,972,785 B2 67 - Continued Thr Glin Gly Ile Asn Glin Gly Pro Glin Ile Asp Llys Glu Gln His Asp 34 O 345 35. O Lys Ile Lieu. Asp Lieu. Ile Glu Ser Gly Lys Lys Glu Gly Ala Lys Lieu. 355 360 365 Glu Cys Gly Gly Gly Arg Trp Gly Asn Lys Gly Phe Phe Val Glin Pro 37 O 375 38O Thr Val Phe Ser Asn Val Thr Asp Glu Met Arg Ile Ala Lys Glu Glu 385 390 395 4 OO Ile Phe Gly Pro Val Glin Glin Ile Met Llys Phe Lys Ser Ile Asp Asp 4 OS 41O 415 Val Ile Lys Arg Ala Asn. Asn. Thir Thr Tyr Gly Lieu Ala Ala Gly Val 42O 425 43 O Phe Thir Lys Asp Lieu. Asp Arg Ala Ile Thr Val Ser Ser Ala Lieu. Glin 435 44 O 445 Ala Gly Val Val Trp Val Asn Cys Tyr Met Ile Leu Ser Ala Glin Cys 450 45.5 460 Pro Phe Gly Gly Phe Lys Met Ser Gly Asin Gly Arg Glu Lieu. Gly Glu 465 470 47s 48O His Gly Lieu. Tyr Glu Tyr Thr Glu Lieu Lys Thr Val Ala Met Lys Ile 485 490 495 Ser Glin Lys Asn. Ser SOO

<210s, SEQ ID NO 10 &211s LENGTH: 2O4 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 10

Met Ala Glu Glin Glu Pro Thir Ala Glu Glin Lieu. Ala Glin Ile Ala Ala 1. 5 1O 15 Glu Asn. Glu Glu Asp Glu. His Ser Val Asn Tyr Llys Pro Pro Ala Glin 2O 25 3O Llys Ser Ile Glin Glu Ile Glin Glu Lieu. Asp Lys Asp Asp Glu Ser Lieu. 35 4 O 45 Arg Llys Tyr Lys Glu Ala Lieu. Lieu. Gly Arg Val Ala Val Ser Ala Asp SO 55 6 O Pro Asn Val Pro Asn Val Ile Val Thr Arg Lieu. Thir Lieu Val Cys Ser 65 70 7s 8O Thir Ala Pro Gly Pro Lieu. Glu Lieu. Asp Lieu. Thr Gly Asp Lieu. Glu Ser 85 90 95 Phe Llys Lys Glin Ser Phe Val Lieu Lys Glu Gly Val Glu Tyr Arg Ile 1OO 105 11 O Lys Ile Ser Phe Arg Val Asn Arg Glu Ile Val Ser Gly Met Lys Tyr 115 12 O 125 Ile Glin His Thr Tyr Arg Lys Gly Val Lys Ile Asp Llys Thr Asp Tyr 13 O 135 14 O Met Val Gly Ser Tyr Gly Pro Arg Ala Glu Glu Tyr Glu Phe Lieu. Thr 145 150 155 160 Pro Met Glu Glu Ala Pro Llys Gly Met Lieu Ala Arg Gly Ser Tyr Asn 1.65 17O 17s Ile Llys Ser Arg Phe Thr Asp Asp Asp Llys Thr Asp His Lieu. Ser Trp 18O 185 19 O Glu Trp Asn Lieu. Thir Ile Llys Lys Glu Trp Lys Asp 195 2OO US 7,972,785 B2 69 70 - Continued

<210s, SEQ ID NO 11 &211s LENGTH: 261 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 11

Met Ala Ser Ala Asp Trp Gly Asp Ser Lys Asn Gly Pro Asp Glin 1. 5 1O 15

Trp Ser Lys Lieu. Tyr Pro Ile Ala Asn Gly ASn Asn Glin Ser Pro Ile 2O 25

Asp Ile Llys Thir Ser Glu Ala Lys His Asp Ser Ser Lell Pro Wall 35 4 O 45

Ser Val Ser Tyr Asn Pro Ala Thir Ala Glu Ile Wall Asn Wall Gly SO 55 6 O

His Ser Phe His Wal Wall Phe Asp Asp Ser Ser Asn Glin Ser Wall Luell 65 70

Lys Gly Gly Pro Lieu Ala Asp Ser Arg Luell Thir Glin Phe His Phe 85 90 95

His Trp Gly Asn. Ser Asn Asp His Gly Ser Glu His Thir Wall Asp Gly 1OO 105 11 O

Ala Lys Tyr Ser Gly Glu Lieu His Luell Wall His Trp Asn Ser Ala 115 12 O 125

Tyr Ser Ser Ala Ala Glu Ala Ile Ser Ala Asp Gly Luell Ala Ile 13 O 135 14 O

Ile Gly Val Lieu Met Llys Val Gly Pro Ala ASn Pro Asn Luell Glin Lys 145 150 155 160

Val Lieu. Asp Ala Lieu. Ser Ser Wall Thir Gly Arg Ala Pro 1.65

Phe Thr Asn Phe Asp Pro Ser Ser Luell Luell Pro Ser Ser Luell Asp Tyr 18O 185 19 O

Trp. Thir Tyr Phe Gly Ser Lieu. Thir His Pro Pro Lell His Glu Ser Wall 195 2O5

Thir Trp Val Ile Cys Lys Glu Ser Ile Ser Luell Ser Pro Glu Glin Luell 21 O 215 22O

Ala Glin Lieu. Arg Gly Lieu. Lieu Ser Ser Ala Glu Gly Glu Pro Ala Wall 225 23 O 235 24 O

Pro Val Lieu. Ser Asn His Arg Pro Pro Glin Pro Lell Gly Arg Thir 245 250 255 Val Arg Ala Ser Phe 26 O

<210s, SEQ ID NO 12 &211s LENGTH: 260 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 12

Met Ser His His Trp Gly Tyr Ser Ser ASn Gly Pro Glu Asn Trp 1. 5 1O 15

His Lys Glu Phe Pro Ile Ala Asn Gly Asp Arg Glin Ser Pro Wall Asp 2O 25 3O

Ile Asp Thr Gly Thr Ala Glin His Asp Pro Ser Lell Glin Pro Luell Luell 35 4 O 45

Ile Cys Tyr Asp Llys Val Ala Ser Ser Ile Wall Asn Asn Gly His SO 55 6 O

Ser Phe Asin Val Glu Phe Asp Asp Ser Glin Asp Phe Ala Wall Luell US 7,972,785 B2 71 72 - Continued

65 70

Glu Gly Pro Luell Ser Gly Ser Arg Luell Ile Glin Phe His Phe His 85 90 95

Trp Gly Ser Ser Asp Gly Glin Gly Ser Glu His Thir Wall Asn 1OO 105 11 O

Ala Ala Glu Lell His Luell Wall His Trp Asn Thir Gly 115 12 O 125

Asp Phe Gly Lys Ala Wall Glin His Pro Asp Gly Lell Ala Wall Luell Gly 13 O 135 14 O

Ile Phe Luell Lys Ile Gly Pro Ala Ser Glin Gly Lell Glin Ile Thir 145 150 155 160

Glu Ala Luell His Ser Ile Thir Gly Lys Arg Ala Ala Phe Ala 1.65 17O 17s

Asn Phe Asp Pro Cys Ser Lell Luell Pro Gly ASn Lell Asp Tyr Trp Thir 18O 185 19 O

Pro Gly Ser Luell Thir Thir Pro Pro Luell Luell Glu Cys Wall Thir Trp 195

Ile Wall Luell Lys Glu Pro Ile Thir Wall Ser Ser Glu Glin Met Ser His 21 O 215

Phe Arg Luell Asn Phe Asn Ser Glu Gly Glu Ala Glu Glu Luell Met 225 23 O 235 24 O

Wall Asp Asn Trp Arg Pro Ala Glin Pro Luell Lys Asn Arg Ile Lys 245 250 255

Ala Ser Phe Lys 26 O

<210s, SEQ ID NO 13 &211s LENGTH: 490 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 13

Met Asp Pro Val Val Wall Lell Luell Luell Ser Luell Phe Phe Luell Luell Phe 1. 5 1O 15

Lell Ser Luell Trp Arg Lell Ser Ser Gly Arg Gly Lell Pro Pro Gly 2O 25

Pro Thir Pro Leul Pro Ile Ile Gly Asn Phe Phe Glin Wall Asp Met Lys 35 4 O 45

Asp Ile Arg Glin Ser Lell Thir Asn Phe Ser Thir Gly Pro Wall SO 55 6 O

Tyr Thir Luell Tyr Val Gly Ser Glin Pro Thir Wall Wall Lell His Gly Tyr 65 70

Glu Ala Luell Lys Glu Ala Lell Wall Asp His Gly Glu Glu Phe Ser Gly 85 90 95

Arg Gly Arg Leul Pro Ile Glu Lys Wall Ala Gly Glin Gly Ile 1OO 105 11 O

Ala Phe Ser His Gly Asn Wall Trp Ala Thir Arg His Phe Thir Wall 115 12 O 125

Thir Luell Arg Asn Lell Gly Met Gly Gly Thir Ile Glu Asp 13 O 135 14 O

Wall Glin Glu Glu Ala Lys Trp Luell Wall Glu Lell Thir Asn 145 150 155 160

Gly Ser Pro Pro Glin Phe Ile Met Gly Ala Pro Gly Asn 1.65 17O 17s

Wall Ile Cys Ile Ile Lell Glin Asn Arg Phe Asp Glu Asp US 7,972,785 B2 73 74 - Continued

18O 185 19 O

Asp Phe Luell Asn Lell Ile Glu Lys Wall Asn Glu Ala Wall Lys Ile Ile 195 2O5

Ser Ser Pro Gly Ile Glin Wall Phe Asn Ile Phe Pro Ile Luell Luell Asp 21 O 215 22O

Tyr Pro Gly Asn His Asn Ile Luell Lys Asn Thir Trp Wall 225 23 O 235 24 O

Ser Luell Lell Glu Ile Glu His Glu Glu Ser Luell Asp 245 250 255

Wall Ser Asn Pro Arg Asp Phe Ile Asp Phe Lell Ile Glu Arg Asn 26 O 265 27 O

Glin Glu Asn Ala Asn Glin Trp Met Asn Thir Lell Glu His Luell Ala 27s 285

Ile Met Wall Thir Asp Lell Phe Phe Ala Gly Ile Glu Thir Wall Ser Ser 29 O 295 3 OO

Thir Met Arg Phe Ala Lell Lell Luell Luell Met Lys Pro His Wall Thir 3. OS 310 315

Ala Wall Glin Glu Glu Ile Asp His Wall Ile Gly Arg His Arg Ser 3.25 330 335

Pro Ser Met Glin Asp Arg Ser His Met Pro Thir Asn Ala Met Wall 34 O 345 35. O

His Glu Wall Glin Arg Ile Asp Ile Gly Pro Asn Gly Luell Luell His 355 360 365

Asp Wall Thir Asp Thir Lys Phe Arg Asn Phe Ile Pro Gly 37 O 375

Thir Ala Wall Luell Thir Ser Lell Thir Ser Wall Luell His Asp Ser Glu 385 390 395 4 OO

Phe Pro Asn Pro Glu Met Phe Asp Pro Gly His Phe Lell Asp Glu Asn 4 OS 415

Gly Asn Phe Lys Lys Ser Asp Phe Ile Pro Phe Ser Ala Gly 425 43 O

Arg Met Cys Luell Gly Glu Ser Luell Ala Arg Met Glu Lell Phe Luell Phe 435 44 O 445

Lell Thir Thir Ile Lell Glin Asn Phe Luell Ser Lell Wall Asp Pro 450 45.5 460

Lys Asp Ile Asn Thir Thir Pro Ile Ser Ser Lell Ser Ser Wall Pro 465 470 47s 48O

Pro Thir Phe Glin Met Arg Phe Ile Pro Luell 485 490

<210s, SEQ ID NO 14 &211s LENGTH: 609 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 14

Met Ser Glu Trp Glu Ser Thir Glu Gly Glu Glu Glu Glu 1. 5 1O 15

Glu Glu Glu Glin Ser Pro Asp Thir Asn Gly Glu Tyr Ser Gly 2O 25 3O

Arg Asp Ser Luell Ile Phe Lell Wall Asp Ala Ser Arg Ala Met Phe Glu 35 4 O 45

Ser Glin Gly Glu Asp Glu Lell Thir Pro Phe Asp Met Ser Ile Glin SO 55 6 O

Ile Glin Ser Wall Tyr Thir Ser Ile Ile Ser Ser Asp Arg Asp Luell US 7,972,785 B2 75 76 - Continued

65 70

Lell Ala Wall Wall Phe Gly Thir Glu Lys Asp Asn Ser Wall Asn 85 90 95

Phe Ser Ile Tyr Wall Lell Glin Asp Luell Asp Asn Pro Gly Ala 1OO 105 11 O

Arg Wall Luell Glu Lell Asp Arg Phe Gly Glin Glin Gly His 115 12 O 125

Phe Arg Asp Thir Ile Gly His Gly Ser Asp Ser Lell Ser Glu Wall 13 O 135 14 O

Lell Trp Wall Ala Asn Lell Phe Ser Asp Wall Glin Phe Met Ser 145 150 155 160

His Arg Ile Met Lell Phe Thir Asn Glu Asp Asp Pro His Gly Asn 1.65 17O 17s

Asp Ser Ala Lys Ala Ser Arg Ala Arg Thir Ala Ser Asp Luell Arg 18O 185 19 O

Asp Thir Gly Ile Phe Lell Asp Luell Met His Luell Lys Arg Gly Gly 195

Phe Asp Wall Ser Lell Phe Tyr Arg Asp Ile Ile Ser Ile Ala Glu Asp 21 O 215 22O

Glu Asp Luell Gly Wall His Phe Glu Glu Ser Ser Lell Glu Asp Luell 225 23 O 235 24 O

Lell Arg Wall Arg Ala Glu Thir Lys Arg Wall Luell Ser Arg 245 250 255

Lell Phe Lys Lell Gly Asp Wall Ala Luell Met Wall Gly Wall Tyr 26 O 265 27 O

Asn Luell Wall Glin Ala Asn Lys Pro Phe Pro Wall Arg Luell Tyr 285

Glu Thir Asn Glu Pro Wall Lys Thir Thir Arg Thir Phe Asn Wall Asn 29 O 295 3 OO

Thir Gly Ser Luell Lell Lell Pro Ser Thir Lys Arg Ser Luell Thir Phe 3. OS 310 315

Gly Thir Arg Glin Ile Wall Lell Glu Glu Glu Thir Glu Glu Luell 3.25 330 335

Arg Phe Asp Glu Pro Gly Lell Ile Luell Met Gly Phe Pro Met Wall 34 O 345 35. O

Met Luell Lys Asn His His Luell Arg Pro Ser Lell Phe Luell Tyr Pro 355 360 365

Glu Glu Ser Luell Wall Asn Gly Ser Ser Thir Luell Phe Ser Ala Luell Luell 37 O 375

Thir Wall Glu Lys Glu Wall Ile Ala Wall Arg Thir Ala 385 390 395 4 OO

Arg Asn Wall Ser Pro Phe Wall Ala Luell Wall Pro Glin Glu Glu 4 OS 415

Glu Luell Asp Asp Glin Asn Ile Glin Wall Thir Pro Ala Gly Phe Glin Luell 425 43 O

Wall Phe Luell Pro Tyr Ala Asp Asp Arg Wall Pro Phe Thir Glu 435 44 O 445

Wall Met Ala Asn Pro Glu Glin Ile Asp Met Ala His Wall 450 45.5 460

Glin Luell Arg Phe Thir Arg Ser Asp Ser Phe Glu Asn Pro Wall 465 470 47s

Lell Glin Glin His Phe Arg Asn Luell Glu Ala Luell Ala Lell Asp Met Met 485 490 495 US 7,972,785 B2 77 - Continued Glu Ser Glu Glin Val Val Asp Lieu. Thir Lieu Pro Llys Val Glu Ala Ile SOO 505 51O Llys Lys Arg Lieu. Gly Ser Lieu Ala Asp Glu Phe Lys Glu Lieu Val Tyr 515 52O 525 Pro Pro Gly Tyr Asn Pro Glu Gly Lys Ile Ala Lys Arg Lys Ala Asp 53 O 535 54 O Asn Glu Gly Ser Ala Ser Llys Llys Pro Llys Val Glu Lieu. Ser Glu Glu 5.45 550 555 560 Glu Lieu Lys Asp Lieu. Phe Ala Lys Gly. Thir Lieu. Gly Llys Lieu. Thr Val 565 st O sts Pro Ala Lieu. Arg Asp Ile Cys Lys Ala Tyr Gly Lieu Lys Ser Gly Pro 58O 585 59 O Llys Lys Glin Glu Lieu. Lieu. Glu Ala Lieu. Ser Arg His Lieu. Glu Lys Thr 595 6OO 605 Asp

<210s, SEQ ID NO 15 &211s LENGTH: 1961 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 15 Met Ala Glin Glin Ala Ala Asp Llys Tyr Lieu. Tyr Val Asp Lys Asn. Phe 1. 5 1O 15 Ile Asin Asn Pro Lieu Ala Glin Ala Asp Cys Gly Ala Lys Llys Lieu Val 2O 25 3O Trp Val Pro Ser Thr Lys Asn Gly Phe Glu Pro Ala Ser Leu Lys Glu 35 4 O 45 Glu Val Gly Glu Glu Ala Ile Val Glu Lieu Val Glu Asn Gly Llys Llys SO 55 6 O Val Llys Val Asn Lys Asp Asp Ile Glin Llys Met Asn Pro Pro Llys Phe 65 70 7s 8O Ser Llys Val Glu Asp Met Ala Glu Lieu. Thir Cys Lieu. Asn. Glu Ala Ser 85 90 95 Val Lieu. His Asn Lieu Lys Glu Arg Tyr Tyr Ser Gly Lieu. Ile Tyr Thr 1OO 105 11 O Tyr Ser Gly Lieu Phe Cys Val Val Ile Asin Pro Tyr Lys Asn Lieu Pro 115 12 O 125 Ile Tyr Ser Glu Glu Ile Val Asp Met Tyr Lys Gly Llys Lys Arg His 13 O 135 14 O Glu Met Pro Pro His Ile Tyr Ala Ile Thr Asp Thr Ala Tyr Arg Ser 145 150 155 160 Met Met Glin Asp Arg Glu Asp Glin Ser Ile Lieu. Cys Thr Gly Glu Ser 1.65 17O 17s Gly Ala Gly Lys Thr Glu Asn. Thir Lys Llys Val Ile Glin Tyr Lieu Ala 18O 185 19 O His Val Ala Ser Ser His Llys Ser Lys Lys Asp Glin Gly Glu Lieu. Glu 195 2OO 2O5 Arg Glin Lieu. Lieu. Glin Ala Asn Pro Ile Lieu. Glu Ala Phe Gly Asn Ala 21 O 215 22O Llys Thr Val Lys Asn Asp Asn. Ser Ser Arg Phe Gly Llys Phe Ile Arg 225 23 O 235 24 O Ile Asin Phe Asp Wall Asn Gly Tyr Ile Val Gly Ala Asn. Ile Glu Thr 245 250 255

Tyr Lieu. Lieu. Glu Lys Ser Arg Ala Ile Arg Glin Ala Lys Glu Glu Arg US 7,972,785 B2 79 80 - Continued

26 O 265 27 O

Thir Phe His Ile Phe Luell Luell Ser Gly Ala Gly Glu His Luell 27s 285

Thir Asp Luell Lell Lell Glu Pro Asn Lys Tyr Arg Phe Luell Ser 29 O 295 3 OO

Asn Gly His Wall Thir Ile Pro Gly Glin Glin Asp Asp Met Phe Glin 3. OS 310 315

Glu Thir Met Glu Ala Met Arg Ile Met Gly Ile Pro Glu Asp Glu Glin 3.25 330 335

Met Gly Luell Luell Arg Wall Ile Ser Gly Wall Luell Glin Lell Gly Asn Ile 34 O 345 35. O

Wall Phe Lys Glu Arg Asn Thir Asp Glin Ala Ser Met Pro Asp Asn 355 360 365

Thir Ala Ala Glin Wall Ser His Luell Luell Gly Ile Asn Wall Thir Asp 37 O 375

Phe Thir Arg Gly Ile Lell Thir Pro Arg Ile Lys Wall Gly Arg Asp Tyr 385 390 395 4 OO

Wall Glin Ala Glin Thir Glu Glin Ala Asp Phe Ala Ile Glu Ala 4 OS 415

Lell Ala Ala Thir Glu Arg Met Phe Arg Trp Lell Wall Luell Arg 425 43 O

Ile Asn Lys Ala Lell Asp Thir Glin Gly Ala Ser Phe Ile 435 44 O 445

Gly Ile Luell Asp Ile Ala Gly Phe Glu Ile Phe Asp Lell Asn Ser Phe 450 45.5 460

Glu Glin Luell Ile Asn Tyr Thir Asn Glu Lys Lell Glin Glin Luell Phe 465 470

Asn His Thir Met Phe Ile Lell Glu Glin Glu Glu Glin Arg Glu Gly 485 490 495

Ile Glu Trp Asn Phe Ile Asp Phe Gly Luell Asp Lell Glin Pro Ile SOO 505

Asp Luell Ile Glu Pro Ala Gly Pro Pro Gly Ile Lell Ala Luell Luell 515 525

Asp Glu Glu Trp Phe Pro Ala Thir Asp Lys Ser Phe Wall Glu 53 O 535 54 O

Lys Wall Wall Glin Glu Glin Gly Thir His Pro Lys Phe Glin Pro Lys 5.45 550 555 560

Glin Luell Asp Lys Ala Asp Phe Ile Ile His Ala Gly Lys 565 st O sts

Wall Asp Lys Ala Asp Glu Trp Luell Met Asn Met Asp Pro Luell 585 59 O

Asn Asp Asn Ile Ala Thir Lell Luell His Glin Ser Ser Asp Phe Wall 595 605

Ser Glu Luell Trp Asp Wall Asp Arg Ile Ile Gly Lell Asp Glin Wall 610 615

Ala Gly Met Ser Glu Thir Ala Luell Pro Gly Ala Phe Thir Arg Lys 625 630 635 64 O

Gly Met Phe Arg Thir Wall Gly Glin Luell Tyr Glu Glin Luell Ala Lys 645 650 655

Lell Met Ala Thir Lell Arg Asn Thir Asn Pro ASn Phe Wall Cys Ile 660 665 67 O

Ile Pro Asn His Glu Ala Gly Luell Asp Pro His Luell Wall 675 68O 685 US 7,972,785 B2 81 - Continued Lieu. Asp Gln Lieu. Arg Cys Asn Gly Val Lieu. Glu Gly Ile Arg Ile Cys 69 O. 695 7 OO Arg Glin Gly Phe Pro Asn Arg Val Val Phe Glin Glu Phe Arg Glin Arg 7 Os 71O 71s 72O Tyr Glu Ile Lieu. Thr Pro Asn Ser Ile Pro Lys Gly Phe Met Asp Gly 72 73 O 73 Lys Glin Ala Cys Val Lieu Met Ile Lys Ala Lieu. Glu Lieu. Asp Ser Asn 740 74. 7 O Lieu. Tyr Arg Ile Gly Glin Ser Llys Val Phe Phe Arg Ser Gly Val Lieu. 7ss 760 765 Ala His Lieu. Glu Glu Glu Arg Asp Lieu Lys Ile Thr Asp Val Ile Ile 770 775 78O Gly Phe Glin Ala Cys Cys Arg Gly Tyr Lieu Ala Arg Lys Ala Phe Ala 78s 79 O 79. 8OO Lys Arg Glin Glin Glin Lieu. Thir Ala Met Llys Val Lieu. Glin Arg Asn. Cys 805 810 815 Ala Ala Tyr Lieu. Arg Lieu. Arg Asn Trp Glin Trp Trp Arg Lieu. Phe Thr 82O 825 83 O Llys Val Llys Pro Lieu. Lieu. Asn. Ser Ile Arg His Glu Asp Glu Lieu. Lieu. 835 84 O 845 Ala Lys Glu Ala Glu Lieu. Thir Lys Val Arg Glu Lys His Lieu Ala Ala 850 855 860 Glu Asn Arg Lieu. Thr Glu Met Glu Thr Met Glin Ser Gln Leu Met Ala 865 87O 87s 88O Glu Lys Lieu Gln Lieu. Glin Glu Gln Lieu. Glin Ala Lys Thr Glu Lieu. Cys 885 890 895 Ala Glu Ala Glu Glu Lieu. Arg Ala Arg Lieu. Thir Ala Lys Lys Glin Glu 9 OO 905 91 O Lieu. Glu Glu Ile Cys His Asp Lieu. Glu Ala Arg Val Glu Glu Glu Glu 915 92 O 925 Glu Arg Cys Glin Tyr Lieu. Glin Ala Glu Lys Llys Llys Met Glin Glin Asn 93 O 935 94 O Ile Glin Glu Lieu. Glu Glu Gln Lieu. Glu Glu Glu Glu Ser Ala Arg Glin 945 950 955 96.O Llys Lieu. Glin Lieu. Glu Lys Val Thir Thr Glu Ala Lys Lieu Lys Llys Lieu. 965 97O 97. Glu Glu Asp Glin Ile Ile Met Glu Asp Glin Asn. Cys Llys Lieu Ala Lys 98O 985 99 O Glu Lys Llys Lieu. Lieu. Glu Asp Arg Val Ala Glu Phe Thir Thir Asp Lieu. 995 1OOO 1005 Met Glu Glu Glu Glu Lys Ser Lys Ser Lieu Ala Lys Lieu Lys Asn O1O O15 O2O Llys His Glu Ala Met Ile Thr Asp Lieu. Glu Glu Arg Lieu. Arg Arg O25 O3 O O35 Glu Glu Lys Glin Arg Glin Glu Lieu. Glu Lys Thr Arg Arg Llys Lieu. O4 O O45 OSO Glu Gly Asp Ser Thr Asp Lieu. Ser Asp Glin Ile Ala Glu Lieu. Glin O55 O6 O O65 Ala Glin Ile Ala Glu Lieu Lys Met Glin Lieu Ala Lys Lys Glu Glu Of O O7 O8O Glu Lieu. Glin Ala Ala Lieu Ala Arg Val Glu Glu Glu Ala Ala Glin O85 O9 O O95

Lys Asn Met Ala Lieu Lys Lys Ile Arg Glu Lieu. Glu Thr Glin Ile 1 OO 105 11 O US 7,972,785 B2 84 - Continued

Ser Lell Glin Glu Asp Glu Ser Glu Arg Ala Cys Arg Asn 25

Glu Glin Lys Asp Luell Gly Glu Luell Glu Ala

Lell Thir Glu Luell Glu Thir Luell Asp Ser Ala Ala Glin

Glin Lell Arg Ser Lys Glu Glin Glu Wall Ile Lell

Lell Glu Asp Glu Thir His Glu Glin Ile Glin

Glu Arg Glin His Glin Ala Wall Glu Luell Ala Glu

Glin Luell Glu Glin Thir Lys Wall Ala Thir Lell Glu Ala 2O5 215

Glin Thir Lell Glu Asn Arg Gly Glu Lell Ala ASn Glu Wall 22O 23 O

Lell Lell Glin Gly Gly Asp Ser Glu Arg

Glu Ala Glin Luell Glin Glu Luell Glin Wall Phe Ser Glu 255

Gly Arg Wall Arg Thir Glu Luell Ala Asp Wall Ser Lell 27 O 27s

Glin Glu Lell Asp Ser Thir Gly Luell Lell Asn Glin Ser Asp 285 29 O

Ser Ser Ser Luell Asp Phe Ser Ala Luell Glu Ser 305

Glin Luell Glin Asp Thir Glin Glu Luell Luell Glin Glu Glu ASn Arg Glin 310 315 32O

Luell Ser Lell Ser Thir Luell Glin Met Glu Asp Glu 3.25 33 O 335

Asn Ser Phe Arg Glu Glin Luell Glu Glu Glu Glu Glu Glu Ala 34 O 345 350

Arg Asn Lell Glu Glin Ala Thir Luell His Ala Glin Wall Thir 355 365

Asp Met Met Asp Gly Wall Gly Luell Glu Thir 37O

Ala Glu Glu Ala Luell Glin Asp Glu Gly Lell 385

Ser Glin Arg Lell Glu Glu Wall Ala Ala Lell Glu 4 OO

Thir Arg Luell Glin Glin Glu Luell Asp Luell Lell Wall 415 42O

Asp Luell His Glin Arg Glin Ser Wall Ser Asn Glu 43 O 435

Glin Phe Asp Glin Luell Luell Ala Glu Glu Thir Ile Ser 450

Ala Ala Glu Glu Asp Arg Ala Glu Ala Glu Ala Arg 465 47 O

Glu Glu Thir Ala Luell Ser Luell Ala Arg Ala Luell Glu Glu 48O 485

Ala Met Glu Glin Ala Glu Luell Glu Arg Lell Asn Glin Phe 490 495 SOO

Arg Glu Met Glu Asp Luell Met Ser Ser Asp Asp Wall Gly US 7,972,785 B2 85 - Continued

5 OS 510 515 Llys Ser Val His Glu Lieu. Glu Lys Ser Asn Arg Ala Lieu. Glu Glin 52O 525 53 O Glin Val Glu Glu Met Lys Thr Glin Lieu. Glu Glu Lieu. Glu Asp Glu 535 54 O 545 Lieu. Glin Ala Thr Glu Asp Ala Lys Lieu. Arg Lieu. Glu Val Asn Lieu. 550 555 560 Glin Ala Met Lys Ala Glin Phe Glu Arg Asp Lieu. Glin Gly Arg Asp 565 st O sts Glu Glin Ser Glu Glu Lys Llys Lys Glin Lieu Val Arg Glin Val Arg 58O 585 590 Glu Met Glu Ala Glu Lieu. Glu Asp Glu Arg Lys Glin Arg Ser Ile 595 6OO 605 Ala Met Ala Ala Arg Llys Llys Lieu. Glu Met Asp Lieu Lys Asp Lieu. 610 615 62O Glu Ala His Ile Asp Thir Ala Asn Lys Asn Arg Glu Glu Ala Ile 625 63 O 635 Lys Glin Lieu. Arg Llys Lieu. Glin Ala Glin Met Lys Asp Cys Met Arg 64 O 645 650 Asp Wall Asp Asp Thir Arg Ala Ser Arg Glu Glu Ile Lieu Ala Glin 655 660 665 Ala Lys Glu Asn. Glu Lys Llys Lieu Lys Ser Met Glu Ala Glu Met 670 675 68O Ile Glin Lieu. Glin Glu Glu Lieu Ala Ala Ala Glu Arg Ala Lys Arg 685 69 O. 695 Glin Ala Glin Glin Glu Arg Asp Glu Lieu Ala Asp Glu Ile Ala Asn 7 OO 7Os 71O Ser Ser Gly Lys Gly Ala Lieu Ala Lieu. Glu Glu Lys Arg Arg Lieu. 71s 72 O 72

Glu Ala Lieu. Ile Ala Lieu. Lieu. Glu Glu Glu Lieu. Glu Glu Glu Glin 73 O 73 74 O Gly Asn Thr Glu Lieu. Ile Asn Asp Arg Lieu Lys Lys Ala Asn Lieu. 74. 7 O 7ss Glin Ile Asp Glin Ile Asn. Thir Asp Lieu. Asn Lieu. Glu Arg Ser His 760 765 770 Ala Glin Lys Asn. Glu Asn Ala Arg Glin Gln Lieu. Glu Arg Glin Asn 775 78O 78s Lys Glu Lieu Lys Ala Lys Lieu. Glin Glu Met Glu Ser Ala Wall Lys 79 O 79. 8OO Ser Lys Tyr Lys Ala Ser Ile Ala Ala Lieu. Glu Ala Lys Ile Ala 805 810 815 Glin Lieu. Glu Glu Gln Lieu. Asp Asn. Glu Thir Lys Glu Arg Glin Ala 82O 825 83 O Ala Ser Lys Glin Val Arg Arg Ala Glu Lys Llys Lieu Lys Asp Wall 835 84 O 845 Lieu. Lieu. Glin Val Glu Asp Glu Arg Arg Asn Ala Glu Glin Phe Llys 850 855 86 O Asp Glin Ala Asp Lys Ala Ser Thr Arg Lieu Lys Glin Lieu Lys Arg 865 87 O 87s Glin Lieu. Glu Glu Ala Glu Glu Glu Ala Glin Arg Ala Asn Ala Ser 88O 885 890 Arg Arg Llys Lieu. Glin Arg Glu Lieu. Glu Asp Ala Thr Glu Thir Ala 895 9 OO 905 US 7,972,785 B2 87 - Continued Asp Ala Met Asn Arg Glu Wall Ser Ser Lieu Lys Asn Llys Lieu. Arg 1910 1915 1920

Arg Gly Asp Met Pro Phe Wall Wall Thir Arg Arg Ile Val Arg Llys 1925 1930 1935

Gly Thir Gly Asp Cys Ser Asp Glu Glu Val Asp Gly Lys Ala Asp 1940 1945 1950

Gly Ala Asp Ala Lys Ala Thir Glu 1955 1960

SEQ ID NO 16 LENGTH: 151 TYPE PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 16

Met Cys Asp Phe Thir Glu Asp Glin Thir Ala Glu Phe Glu Ala Phe 1. 5 15

Glin Luell Phe Asp Arg Thir Gly Asp Gly Ile Lell Ser Glin Cys 25

Gly Asp Wall Met Arg Ala Lieu. Gly Glin Asn Pro Thir Asn Ala Glu Wall 35 4 O 45

Lell Lys Wall Lieu. Gly Asn Pro Ser Asp Met Asn. Wall Wall SO 55 6 O

Lell Asp Phe Glu His Phe Lell Pro Met Luell Thir Wall Ala Asn 65 70

Asp Glin Gly Thir Glu Asp Wall Gly Lieu. Arg Wall Phe 85 90 95

Asp Glu Gly Asn Gly Thir Wall Met Gly Glu Ile Arg His Val 105 11 O

Lell Wall Thir Lieu. Gly Glu Met Thir Glu Glu Wall Glu Met Lieu. 115 12 O 125

Wall Ala Gly His Glu Asp Ser Asn Gly Asn Glu Glu Lieu. 13 O 135 14 O

Lell Arg Met Wall Lell Asn Gly 145 150

SEO ID NO 17 LENGTH: 554 TYPE PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 17

Met Tyr Ala Leu Ala Lieu. Luell Ala Ser Luell Luell Wall. Thir Ala Lieu. Thir 1. 5 1O 15

Ser Pro Wall Glin Asp Pro Ile Cys Ser Gly Gly Ser Ala Wall Wall 25 3O

Arg Asp Wall Lys Thir Ala Wall Asp Arg Ala Wall His Cys 35 4 O 45

Glin Glin Met Val Trp Ser Lys Pro Thir Ala Ser Leu Pro Asp SO 55 6 O

Ile Thir Wall Wall Thir Glu Ala Gly Lieu. Luell Asp 65 70

Ala Thir Glu Glu Glu Ile Lieu. His Luell Glu Thir Ala Trp 85 90 95

Ile His Asp Ser Ser Leu Ser Ala Ser Glu Wal Wall Asp Ser 105 11 O

Luell Pro Val Ile Lieu. Asp Met Ile Gly Glu Met Ser Asn. Pro US 7,972,785 B2 89 90 - Continued

115 12 O 125

Gly Glu Wall Ser Ala Lell Asn Luell Cys Glin Ser Lell Glin Glu Tyr 13 O 135 14 O

Lell Ala Glu Glin Asn Glin Arg Glin Luell Glu Ser Asn Lys Ile Pro Glu 145 150 155 160

Wall Asp Luell Ala Arg Wall Wall Ala Pro Phe Met Ser Asn Ile Pro Luell 1.65 17O 17s

Lell Luell Pro Glin Asp Arg Pro Arg Ser Glin Pro Glin Pro Ala 18O 185 19 O

Asn Glu Asp Wall Cys Glin Asp Cys Met Luell Wall Thir Asp Ile Glin 195

Thir Ala Wall Arg Thir Asn Ser Ser Phe Wall Glin Gly Lell Wall Asp His 21 O 215 22O

Wall Glu Asp Cys Asp Arg Luell Gly Pro Gly Wall Ser Asp Ile Cys 225 23 O 235 24 O

Asn Wall Asp Glin Ser Glu Wall Ala Wall Glin Met Met Met 245 250 255

His Met Glin Pro Lys Glu Ile Wall Met Wall Gly Phe Cys Asp Glu 26 O 265 27 O

Wall Arg Wall Pro Met Arg Thir Luell Wall Pro Ala Thir Glu Ala Ile 27s 285

Asn Ile Luell Pro Ala Lell Glu Luell Thir Asp Pro Glu Glin Asp 29 O 295 3 OO

Wall Ile Glin Ala Glin Asn Wall Ile Phe Glin Wall Glin Luell Wall 3. OS 310 315

Met Arg Luell Ser Glu Lell Ile Ile Asn ASn Ala Thir Glu Glu Luell 3.25 330 335

Lell Ile Gly Lell Ser Ala Cys Ser Luell Lell Pro Ala Pro Ala 34 O 345 35. O

Ser Thir Lys Glin Glu Wall Luell Wall Thir Phe Gly Pro Ser Luell Luell 355 360 365

Asp Wall Luell Met His Glu Wall Asn Pro Asn Phe Lell Gly Wall Ile 37 O 375

Ser Luell Ser Ala Asn Pro Asn Luell Wall Gly Thir Lell Glu Glin Pro 385 390 395 4 OO

Ala Ala Ala Ile Wall Ser Ala Luell Pro Lys Glu Pro Ala Pro Pro Lys 4 OS 415

Glin Pro Glu Glu Pro Glin Ser Ala Luell Arg Ala His Wall Pro Pro 425 43 O

Glin Asn Gly Gly Phe Glu Wall Lell Wall Ile Tyr 435 44 O 445

Lell Glu His Asn Lell Glu Lys Asn Ser Thir Glu Glu Ile Luell Ala 450 45.5 460

Ala Luell Glu Gly Cys Ser Phe Luell Pro Asp Pro Glin Glin 465 470

Asp Glu Phe Wall Ala Glu Glu Pro Luell Lell Lell Glu Ile Luell 485 490 495

Wall Glu Wall Met Asp Pro Ser Phe Wall Ser Ile Gly Wall Cys SOO 505

Pro Ser Ala Lys Lell Lell Luell Gly Thir Glu Cys Wall Trp Gly 515 52O 525

Pro Gly Trp Cys Glin Asn Ser Glu Thir Ala Ala Arg Asn Ala 53 O 535 54 O US 7,972,785 B2 91 - Continued Val Asp His Cys Lys Arg His Val Trp Asn 5.45 550

<210s, SEQ ID NO 18 &211s LENGTH: 395 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 18 Met Llys Pro Ala Glin Ser Ser Ile Arg Pro Cys Arg Lieu. Arg Pro Pro 1. 5 1O 15 Asn Trp Ala Glu Gly Lys Asp Phe Trp Val Ser Asp Pro Llys Lys Gly 2O 25 3O Glin Ala Arg Asp Gly Thr Ala Lieu Ala Val Ser Arg Ala Gly Ala Lieu. 35 4 O 45 Glin Pro Ser Gly Gly Ala Gly Lieu. Gly Ser Gly Ala Lys Ala Ser His SO 55 6 O Val Arg Pro Ala Ala Lieu Lleu Ser Ser Ile Ser Lieu. Asn Gly Lieu. Arg 65 70 7s 8O Arg Gly Ser Pro Pro Ile Ser Thr Lieu. Trp Asn Ala Gly Arg Gly Glu 85 90 95 Lieu. Glin Asp Arg Arg Asn His Lieu Pro Lys Arg Gly Glu Glu Ala Glu 1OO 105 11 O Ser Glin Phe Lys Arg Arg Arg Glu Lys Asp Glu Glu Ser Gly Trp Pro 115 12 O 125 Ile Glu Ala Ala Glu Asn Gly Arg Glin Ala Ser Phe Arg Phe Ala Ser 13 O 135 14 O Gly Gly Arg Llys Trp Pro Phe Lieu. Gly Gly Trp Llys Val Arg Llys Phe 145 150 155 160 Val Arg Ala Ala Ala Lys Lieu. Arg Arg Asp Ala His Ser Trp Phe Ser 1.65 17O 17s Arg Lieu. Ser Arg Val Ser Gly Ala Gly Arg Arg Ala Gly Val Ala Arg 18O 185 19 O Pro Glin Glu Val Ser Ser Pro Gly Gly Gly Glu Arg Gly Gly Arg Arg 195 2OO 2O5 Glu Pro Arg Lieu. Gly Ser Ala Pro Gly Glin Asp Lieu. Thir Ala Thr Met 21 O 215 22O Ser Ser Lys Arg Ala Lys Thr Lys Thir Thir Lys Lys Arg Pro Glin Arg 225 23 O 235 24 O Ala Thr Ser Asn Val Phe Ala Met Phe Asp Glin Ser Glin Ile Glin Glu 245 250 255 Phe Lys Glu Ala Phe Asn Met Ile Asp Glin Asn Arg Asp Gly Phe Ile 26 O 265 27 O Asp Llys Glu Asp Lieu. His Asp Met Lieu Ala Ser Met Gly Lys Asn Pro 27s 28O 285 Thir Asp Glu Tyr Lieu. Asp Ala Met Met Asn. Glu Ala Pro Gly Pro Ile 29 O 295 3 OO Asn Phe Thr Met Phe Lieu. Thir Met Phe Gly Glu Lys Lieu. Asn Gly Thr 3. OS 310 315 32O Asp Pro Glu Asp Val Ile Arg Asn Ala Phe Ala Cys Phe Asp Glu Glu 3.25 330 335 Ala Ile Gly. Thir Ile Glin Glu Asp Tyr Lieu. Arg Glu Lieu. Lieu. Thir Thr 34 O 345 35. O Met Gly Asp Arg Phe Thr Asp Glu Glu Val Asp Glu Lieu. Tyr Arg Glu 355 360 365 US 7,972,785 B2 93 94 - Continued Ala Pro Ile Asp Llys Lys Gly Asn. Phe Asn Tyr Ile Glu Phe Thr Arg 37 O 375 Ile Lieu Lys His Gly Ala Lys Asp Lys Asp Asp 385 390 395

<210s, SEQ ID NO 19 &211s LENGTH: 928 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 19

Met Gly Asp Met Glin Asp His Glu Wall Luell Glu Ile Pro Asp Arg 1. 5 15

Asp Ser Glu Glu Glu Lell Glu His Wall Ile Glu Glin Ile Ala 25

Asp Luell Asp Ile Pro Wall Thir Glu Met Glin Glu Ser Glu Ala Luell Pro 35 4 O 45

Thir Glu Glin Thir Ala Thir Asp Tyr Ile Pro Thir Ser Thir Ser Thir Ser SO 55 6 O

His Pro Ser Ser Ser Glin Wall Tyr Wall Glu Luell Glin Glu Luell Met Met 65 70

Asp Glin Arg Asn Glin Glu Lell Glin Trp Wall Glu Ala Ala His Trp Ile 85 90 95

Gly Luell Glu Glu Asn Lell Arg Glu Asp Gly Wall Trp Gly Arg Pro His 105 11 O

Lell Ser Tyr Luell Thir Phe Trp Ser Luell Luell Glu Lell Glin Wall Phe 115 12 O 125

Ser Lys Gly Thir Phe Lell Lell Asp Luell Ala Glu Thir Ser Luell Ala Gly 13 O 135 14 O

Wall Ala Asn Lell Lell Asp Ser Phe Ile Tyr Glu Asp Glin Ile Arg 145 150 155 160

Pro Glin Asp Arg Asp Glu Lell Luell Arg Ala Luell Lell Lell Arg Ser 1.65

His Ala Glu Asp Lell Asp Luell Glu Gly Wall Pro Ala Wall Luell 18O 185 19 O

Thir Arg Ser Gly Ala Pro Ser Glu Pro Luell Luell Pro His Glin Pro Ser 195

Lell Glu Thir Lell Cys Ala Glin Ala Glu Gly Gly Ser Glu Glu 21 O 215 22O

Pro Ser Pro Ser Gly Ile Lell Lys Ile Pro Pro Asn Ser Glu Thir Thir 225 23 O 235 24 O

Lell Wall Luell Wall Gly Arg Ala Ser Phe Luell Wall Pro Wall Luell Gly 245 250 255

Phe Wall Arg Luell Lys Glu Ala Wall Pro Luell Glu Asp Lell Wall Luell Pro 26 O 265 27 O

Glu Pro Wall Ser Phe Lell Lell Wall Luell Luell Gly Pro Glu Ala Pro His 285

Ile Asp Thir Glin Lell Gly Arg Ala Ala Ala Thir Lell Met Thir Glu 29 O 295 3 OO

Arg Wall Phe Arg Wall Thir Ala Ser Luell Ala Glin Ser Arg Gly Glu Luell 3. OS 310 315

Lell Ser Ser Luell Asp Ser Phe Luell Asp Cys Ser Lell Wall Luell Pro Pro 3.25 330 335

Thir Glu Ala Pro Ser Glu Ala Luell Luell ASn Lell Wall Pro Wall Glin 34 O 345 35. O US 7,972,785 B2 95 96 - Continued

Glu Luell Luell Arg Arg Tyr Luell Pro Arg Pro Ala Lys Pro Asp 355 360 365

Pro Asn Luell Glu Ala Lell Ala Asp Gly Gly Lys Glu Gly Pro Gly 37 O 375

Asp Glu Asp Asp Pro Lell Arg Arg Thir Gly Arg Ile Phe Gly Gly Luell 385 390 395 4 OO

Ile Arg Asp Ile Arg Arg Arg Pro Tyr Lell Ser Asp Ile Thir 4 OS 415

Asp Ala Luell Ser Pro Glin Wall Luell Ala Ala Wall Ile Phe Ile Phe 425 43 O

Ala Ala Luell Ser Pro Ala Wall Thir Phe Gly Gly Lell Lell Gly Glu 435 44 O 445

Thir Arg Asn Luell Met Gly Wall Ser Glu Luell Luell Ile Ser Thir Ala Wall 450 45.5 460

Glin Gly Ile Luell Phe Ala Lell Luell Gly Ala Glin Pro Lell Luell Wall Luell 465 470

Gly Phe Ser Gly Pro Lell Lell Wall Phe Glu Glu Ala Phe Ser Phe 485 490 495

Glu Ser Asn Asn Lell Glu Tyr Ile Wall Gly Arg Ala Trp Ile Gly SOO 505

Phe Trp Luell Ile Lell Lell Wall Wall Luell Wall Wall Ala Phe Glu Gly Ser 515 525

Phe Luell Wall Glin Tyr Ile Ser Arg Thir Glin Glu Ile Phe Ser Phe 53 O 535 54 O

Lell Ile Ser Luell Ile Phe Ile Glu Thir Phe Ser Luell Ile Lys 5.45 550 555 560

Ile Phe Glin Asp Tyr Pro Lell Glin Glu Ser Ala Pro Wall Wall Met 565 st O sts

Pro Pro Glin Gly Pro Wall Pro Asn Thir Ala Lell Luell Ser Luell 585 59 O

Wall Luell Met Wall Gly Thir Phe Luell Luell Ala Met Met Lell Arg Phe 595 605

Asn Ser Thir Tyr Phe Pro Gly Luell Arg Arg Wall Ile Gly Asp 610 615

Phe Gly Wall Pro Ile Ser Ile Luell Ile Met Wall Lell Wall Asp Thir Phe 625 630 635 64 O

Ile Asn Thir Tyr Thir Glin Luell Ser Wall Pro Asp Gly Luell 645 650 655

Wall Ser Asn Ser Ser Ala Arg Gly Trp Wall Ile His Pro Luell Gly Luell 660 665 67 O

Asn His Phe Pro Trp Met Met Phe Ala Ser Wall Luell Pro Ala 675 68O 685

Lell Luell Wall Phe Ile Lell Ile Phe Luell Glu Ser Glin Ile Thir Thir Luell 69 O. 695 7 OO

Ile Wall Ser Pro Glu Arg Met Ile Lys Gly Ser Gly Phe His 7 Os

Lell Asp Luell Luell Lell Wall Wall Gly Met Gly Gly Wall Ala Ala Luell Phe 72 73 O 73

Gly Met Pro Trp Lell Ser Ala Thir Thir Wall Arg Ser Wall Thir His Ala 740 74. 7 O

Asn Ala Luell Thir Wall Met Gly Lys Ala Ser Gly Pro Gly Ala Ala Ala 760 765

Glin Ile Glin Glu Wall Glu Glin Arg Ile Ser Gly Lell Luell Wall Ser 770 775 78O US 7,972,785 B2 97 98 - Continued

Wall Luell Wall Gly Lell Ser Ile Luell Met Glu Pro Ile Lell Ser Arg Ile 79 O 79.

Pro Luell Ala Wall Lell Phe Gly Ile Phe Luell Tyr Met Gly Ile Thir Ser 805 810 815

Lell Ser Gly Ile Glin Lell Phe Asp Arg Ile Luell Lell Lell Phe Pro 825 83 O

Pro Tyr His Pro Asp Wall Pro Phe Wall Lys Arg Wall Thir Trp 835 84 O 845

Arg Met His Luell Phe Thir Gly Ile Glin Ile Ile Cys Lell Ala Wall Luell 850 855 860

Trp Wall Wall Lys Ser Thir Pro Ala Ser Luell Ala Lell Pro Phe Wall Luell 865

Ile Luell Thir Wall Pro Lell Arg Arg Luell Luell Luell Pro Lell Ile Phe Arg 885 890 895

Glu Luell Glu Luell Glin Lell Asp Gly Asp Asp Ala Wall Thir Phe 9 OO 905 91 O

Asp Glu Ala Glu Gly Lell Asp Glu Tyr Asp Glu Wall Pro Met Pro Wall 915 92 O 925

SEQ ID NO 2 O LENGTH: 199 TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE:

Ala ASn Arg Gly Pro Ser Gly Luell Ser Arg Glu Wall Glin Glin 5 1O 15

Glu Lys Glin Asp Pro Asp Luell Glu Glin Ile Luell Ile Glin 25 3O

Trp Thir Thir Glin Arg Lys Gly Wall Ser Glin Pro Glin Pro Gly 35 4 O 45

Arg Asn Phe Glin Asn Trp Luell Asp Gly Thir Wall Luell Glu 55 6 O

Lell Asn Ser Lell Tyr Pro Glu Gly Glin Ala Pro Wall Ile 65 70

Glin Ser Thir Met Ala Phe Glin Met Glu Glin Ile Ser Glin Phe 85 90 95

Lell Ala Ala Glu Arg Gly Ile Asn Thir Thir Asp Ile Phe Glin 105 11 O

Thir Wall Asp Luell Trp Glu Gly Lys Asn Met Ala Wall Glin Arg Thir 115 12 O 125

Lell Met Asn Luell Gly Gly Lell Ala Wall Ala Arg Asp Asp Gly Luell Phe 13 O 135 14 O

Ser Gly Asp Pro Asn Trp Phe Pro Ser Glu Asn Pro Arg 145 150 155 160

Asn Phe Ser Asp Asn Glin Lell Glin Glu Gly Lys Asn Wall Ile Gly Luell 1.65 17O 17s

Glin Met Gly Thir Asn Arg Gly Ala Ser Glin Ala Gly Met Thir Gly Tyr 18O 185 19 O

Gly Met Pro Arg Glin Ile Lell 195

<210s, SEQ ID NO 21 &211s LENGTH: 247 212. TYPE : PRT <213> ORGANISM: Rattus sp. US 7,972,785 B2 99 100 - Continued

<4 OOs, SEQUENCE: 21 Met Ala Gly Ser Thir Thir Ile Glu Ala Wall Lys Arg Ile Glin Wall 1. 15

Lell Glin Glin Glin Ala Asp Asp Ala Glu Glu Arg Ala Glu Arg Luell Glin 25

Arg Glu Wall Glu Gly Glu Arg Arg Ala Arg Glu Glin Ala Glu Ala Glu 35 4 O 45

Wall Ala Ser Luell Asn Arg Arg Ile Glin Luell Wall Glu Glu Glu Luell Asp SO 55 6 O

Arg Ala Glin Glu Arg Lell Ala Thir Ala Luell Glin Lell Glu Glu Ala 65 70

Glu Ala Ala Asp Glu Ser Glu Arg Gly Met Wall Ile Glu Asn 85 90 95

Arg Ala Luell Lys Asp Glu Glu Met Glu Luell Glin Glu Ile Glin Luell 1OO 105 11 O

Glu Ala His Ile Ala Glu Glu Ala Asp Arg Lys Glu Glu 115 12 O 125

Wall Ala Arg Lell Wall Ile Ile Glu Gly Asp Lell Glu Arg Thir Glu 13 O 135 14 O

Glu Arg Ala Glu Lell Ala Glu Ser Ser Glu Lell Glu Glu Glu 145 150 155 160

Lell Asn Wall Thir Asn Asn Luell Ser Luell Glu Ala Glin Ala Glu 1.65 17O

Ser Glin Lys Glu Asp Tyr Glu Glu Glu Ile Lys Ile Luell 18O 185 19 O

Thir Asp Lys Luell Lys Glu Ala Glu Thir Arg Ala Glu Phe Ala Glu Arg 195 2OO

Ser Wall Ala Lys Lell Glu Lys Thir Ile Asp Asp Lell Glu Glu Arg Luell 21 O 215

Tyr Ser Glin Luell Glu Arg Asn Arg Luell Luell Ser Asn Glu Luell Luell 225 23 O 235 24 O

Thir Luell His Gly Lell Asp 245

<210s, SEQ ID NO 22 &211s LENGTH: 2541 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 22

Met Wall Ala Lieu Ser Lell Arg Ile Ser Ile Gly Asn Wall Wall Lys Thir 1. 5 1O 15

Met Glin Phe Glu Pro Ser Thir Met Wall Asp Ala Arg Met Ile 25

Arg Glu Arg Ile Pro Glu Ala Luell Ala Pro Pro Ser Asp Phe Gly 35 4 O 45

Lell Phe Luell Ser Asp Asp Asp Pro Gly Ile Trp Luell Glu Ala SO 55 6 O

Gly Ala Luell Asp Tyr Met Luell Arg ASn Gly Asp Thir Met Glu 65 70

Glin Arg Pro Luell Ile Arg Met Lell Asp Gly Thir 85 90 95

Wall Thir Ile Met Wall Asp Asp Ser Thir Wall Thir Asp Met Luell 1OO 105 11 O US 7,972,785 B2 101 102 - Continued

Met Thir Ile Ala Arg Ile Gly Ile Thir ASn His Asp Glu Tyr Ser 115 12 O 125

Lell Wall Arg Glu Lell Met Glu Glu Asp Glu Gly Thir Gly Thir 13 O 135 14 O

Lell Arg Asp Lys Thir Lell Luell Arg Asp Glu Lys Met Glu Lys 145 150 155 160

Lell Glin Lell His Thir Asp Asp Glu Luell Asn Trp Luell Asp His 1.65 17O 17s

Gly Arg Thir Luell Arg Glu Glin Gly Wall Glu Glu His Glu Thir Luell Luell 18O 185 19 O

Lell Arg Arg Phe Phe Ser Asp Glin ASn Wall Asp Ser Arg Asp 195

Pro Wall Glin Luell Asn Lell Lell Wall Glin Ala Arg Asp Asp Ile Luell 21 O 215

Asn Gly Ser His Pro Wall Ser Phe Asp Ala Glu Phe Ala Gly 225 23 O 235 24 O

Phe Glin Glin Ile Glin Phe Gly Pro His ASn Glu Glin His 245 250 255

Ala Gly Phe Luell Asp Lell Asp Phe Luell Pro Glu Tyr Wall 26 O 265 27 O

Glin Gly Glu Arg Ile Phe Glin Ala His Asn Gly Glin 28O 285

Met Ser Glu Ile Glu Ala Lys Wall Arg Wall Lys Lell Ala Arg Ser 29 O 295 3 OO

Lell Thir Gly Wall Ser Phe Phe Luell Wall Glu Met Lys 3. OS 310 315

Gly Asn Lell Wall Pro Arg Luell Luell Gly Ile Thir Glu Cys 3.25 330 335

Wall Met Arg Wall Asp Glu Thir Lys Glu Wall Ile Glin Glu Trp Ser 34 O 345 35. O

Lell Thir Asn Ile Lys Arg Trp Ala Ala Ser Pro Ser Phe Thir Luell 355 360 365

Asp Phe Gly Asp Tyr Glin Asp Gly Ser Wall Glin Thir Thir Glu 37 O 375 38O

Gly Glu Glin Ile Ala Glin Lell Ile Ala Gly Tyr Ile Asp Ile Ile Luell 385 390 395 4 OO

Ser Asp His Phe Gly Luell Glu Gly Asp Glu Glu 4 OS 415

Ser Thir Met Luell Glu Asp Ser Wall Ser Pro Lys Ser Thir Wall Luell 425 43 O

Glin Glin Glin Asn Arg Wall Gly Ala Glu His Gly Ser Wall Ala 435 44 O 445

Lell Pro Ala Ile Met Arg Ser Gly Ala Ser Gly Pro Glu Asn Phe Glin 450 45.5 460

Wall Gly Ser Met Pro Pro Ala Glin Glin Glin Wall Thir Ser Gly Glin Met 465 470

His Arg Gly His Met Pro Pro Luell Thir Ser Ala Glin Glin Ala Luell Thir 485 490 495

Gly Thir Ile Asn Ser Ser Met Glin Ala Wall Glin Ala Ala Glin Ala Ala SOO 505

Lell Asp Asp Phe Asp Ala Lell Pro Pro Luell Gly Glin Asp Ala Ala Ser 515 52O 525

Ala Trp Arg Lys Asn Lys Met Asp Glu Ser Lys His Glu Ile His 53 O 535 54 O US 7,972,785 B2 103 104 - Continued

Ser Glin Wall Asp Ala Ile Thir Ala Gly Thir Ala Ser Wal Wall Asn Luell 5.45 550 555 560

Thir Ala Gly Asp Pro Ala Glu Thir Asp Tyr Thir Ala Wall Gly Cys Ala 565 st O sts

Wall Thir Thir Ile Ser Ser Asn Luell Thir Glu Met Ser Arg Gly Wall Lys 585 59 O

Lell Luell Ala Ala Lell Lell Glu Asp Glu Gly Gly Asn Gly Arg Pro Luell 595 605

Lell Glin Ala Ala Lys Gly Lell Ala Gly Ala Wall Ser Glu Luell Luell Arg 610 615

Ser Ala Glin Pro Ala Ser Ala Glu Pro Arg Glin Asn Lell Luell Glin Ala 625 630 635 64 O

Ala Gly Asn Wall Gly Glin Ala Ser Gly Glu Luell Lell Glin Glin Ile Gly 645 650 655

Glu Ser Asp Thir Asp Pro His Phe Glin Asp Ile Lell Met Glin Luell Ala 660 665 67 O

Ala Wall Ala Ser Ala Ala Ala Ala Luell Wall Lell Lys Ala Ser 675 685

Wall Ala Glin Arg Thir Glu Asp Ser Gly Luell Glin Thir Glin Wall Ile Ala 69 O. 695 7 OO

Ala Ala Thir Glin Cys Ala Lell Ser Thir Ser Glin Lell Wall Ala Thir 7 Os

Wall Wall Ala Pro Thir Ile Ser Ser Pro Wall Glin Glu Glin Luell 72 73 O 73

Wall Glu Ala Gly Arg Lell Wall Ala Lys Ala Wall Glu Gly Cys Wall Ser 740 74. 7 O

Ala Ser Glin Ala Ala Thir Glu Asp Gly Glin Luell Lell Arg Gly Wall Gly 760 765

Ala Ala Ala Thir Ala Wall Thir Glin Ala Luell ASn Lell Luell Glin His 770 775

Wall Ala His Ala Thir Gly Ala Gly Pro Ala Arg Asp Glin 79 O 79.

Ala Thir Asp Thir Ile Lell Thir Wall Thir Glu ASn Phe Ser Ser Met 805 810 815

Gly Asp Ala Gly Glu Met Wall Arg Glin Ala Arg Lell Glin Ala 825

Thir Ser Asp Luell Wall Asn Ala Ile Ala Asp Glu Glu Ser 835 84 O 845

Asp Luell Glu Asn Ser Arg Lys Luell Luell Ser Ala Luell Ala 850 855

Asp Ala Thir Ala Lys Met Wall Glu Ala Ala Lys Ala Ala His 865

Pro Asp Ser Glu Glu Glin Glin Glin Arg Luell Arg Ala Glu Gly 885 890 895

Lell Arg Met Ala Thir Asn Ala Ala Ala Glin ASn Ile Lys 9 OO 905

Lell Wall Glin Arg Lell Glu His Ala Ala Glin Ala Ser Ala 915 92 O 925

Thir Glin Thir Ile Ala Ala Ala Glin His Ala Ala Ser Ala Pro Ala 93 O 935 94 O

Ser Ala Gly Pro Glin Pro Lell Luell Wall Glin Ser Ala Wall Ala 945 950 955 96.O

Glu Glin Ile Pro Lell Lell Wall Glin Gly Wall Arg Gly Ser Glin Ala Glin US 7,972,785 B2 105 106 - Continued

965 97.

Pro Asp Ser Pro Ser Ala Glin Lieu Ala Lieu. Ile Ala Ala Ser Glin Ser 98O 985 99 O

Phe Leu Gln Pro Gly Gly Lys Met Wall Ala Ala A a Lys Ala Ser Wall 995 1OOO 1005

Pro Thir Ile Glin Asp Glin Ala Ser Ala Met Glin Lell Ser Glin Cys O15 O2O

Ala Lys Asn Lell Gly Thr Luell Ala Glu Lell Arg Thir Ala Ala O35

Glin Lys Ala Glin Glu Ala Gly Pro Luell Glu Met Asp Ser Ala OSO

Lell Ser Wall Wall Glin Asn Glu Asp Lell Glin Glu Ile O65

Ala Ala Ala Arg Glu Gly Luell Pro Lell Gly Glu Thir

Met Thr Gin Luell Gly ASn Ser Ala Wall

Ser Ser Ala Ile Ala Lys Luell Gly Glu Ile Glin Gly Asn

Glu Ala Gly Ile Ala Arg Asp Wall Gly Gly Lell

Arg Ser Lell Ala Glin Ala Arg Gly Wall Ala Luell Thir Thir

Asp Pro Ala Wall Glin Ala Wall Luell Asp Thir Ser Asp Ala

Lell Ala Ser Ser Ile Glu Glu Ala Ala Ser

Gly His Pro Gly Asp Pro Ser Glin Glin Arg Ala Glin Wall

Ala Lys Ala Wall Thr Gin Luell Asn Arg Wall Ser Lell 2OO

Pro Gly Glin Arg Asp Wall Asn Ala Luell Arg Ala Wall Gly Asp 2O5 215

Ala Ser Lys Arg Lieu. Luell Ser Asp Ser Luell Pro Pro Ser Thir Gly 22O 225 23 O

Thir Phe Glin Glu Ala Glin Ser Arg Luell ASn Glu Ala Ala Ala Gly 235 24 O 245

Lell ASn Glin Ala Ala Thr Glu Luell Wall Glin Ala Arg Gly Thir 250 255

Pro Glin Asp Lell Ala Arg Ser Gly Arg Phe Glin Asp Phe 265

Ser Thir Phe Lell Glu Ala Wall Glu Met Ala Glin Ala Pro 28O

Ser Glin Glu Asp Arg Ala Glin Wall Wall Ser Asn Gly Ile 295 3OO

Ser Met Ser Ser Ser Lys Luell Luell Luell Ala Ala Ala Lell Ser 310 315

Thir Asp Pro Ala Ala Pro Asn Luell Ser Glin Lell Ala Ala Ala 33 O 335

Ala Arg Ala Wall Thir Asp Ser Ile Asn Glin Lell le Thir Met 345 350

Thir Glin Glin Ala Pro Gly Glin Glu Asp Asn Ala Lell Arg 355 360 365 US 7,972,785 B2 107 108 - Continued

Glin Luell Glu Thir Wall Arg Glu Luell Luell Glu Asn Pro Wall Glin Pro 37O 375 38O

Ile Asn Asp Met Ser Tyr Phe Gly Luell Asp Ser Wall Met Glu 385 390 395

Asn Ser Wall Luell Gly Glu Ala Met Thir Gly Ser Glin Asn 4 OO 405

Asn Gly Asn Luell Pro Glu Phe Gly Asp Ile Ala Thir 415

Ser Ala Luell Phe Thir Glu Ala Ala Glin Ala 43 O 435

Lell Wall Gly Wall Ser Asp Pro ASn Ser Ala Gly Glin 445 450

Lell Wall Glu Pro Glin Phe Ala Arg ASn Glin Ala 460 465

Glin Met Ala Glin Ser Luell Gly Glu Pro Thir Glin 47s 48O

Glin Wall Lell Ser Ala Thir Ile Wall Ala His Thir Ser 490

Luell Asn Ser Luell Ala Ser Ala Thir Ala Asn 5 OS

Pro Ala Arg Glin Wall Glin Ser Ala Glu Wall Ala

Asn Ser Thir Ala Asn Luell Lys Thir Ile Ala Luell Asp Gly 535 545

Ala Phe Thir Glu Glu Asn Ala Glin Arg Ala Ala Thir Ala 550 560

Pro Luell Lell Glu Ala Wall Asn Luell Ser Ala Phe Ala Ser Asn 565 sts

Pro Phe Ser Ser Wall Ala Glin Ile Ser Pro Glu Gly Arg 590

Ala Met Glu Pro Ile Ile Ser Ala Met Lell Glu 605

Ser Gly Gly Luell Ile Glin Thir Ala Arg Ala Lell Ala Wall Asn 615 62O

Pro Asp Pro Pro Arg Ser Wall Luell Ala His Ser Arg 63 O

Thir Ser Asp Ser Ile Lys Luell Ile Thir Met Arg Asp 645

Pro Gly Glin Luell Glu Glu Thir Ala Ala Ala Lell 660

Asn Ser Lell Arg Asp Luell Asp Glin Ala Ser Lell Ala Ala Wall 670 675 68O

Ser Glin Glin Lell Ala Pro Glu Gly Ile Ser Glin Glu Ala Lell 685 695

His Glin Met Luell Thir Wall Glin Glu Ile His Lell Ile 7 OO

Glu Lell Ala Ser Ala Arg Ala Glu Ala Glin Lell Gly

His Wall Ser Glin Met Glin Phe Glu Luell Thir Lell

Ala Wall Gly Ala Ala Ser Thir Luell Ser Pro Glin Glin 7 O

Met Lell Lell Asp Glin Thir Luell Ala Glu Ser Ala Lell 765 770 US 7,972,785 B2 109 110 - Continued

Glin Luell Lell Tyr Thir Ala Lys Glu Ala Gly Gly Asn Pro Lys Glin 775 78O 78s

Ala His Thir Glin Glu Ala Luell Glu Glu Ala Wall Glin Met Met 79. 8OO

Thir Ala Wall Glu Asp Luell Thir Thir Thir Lell Asn Glu Ala Ala 810 815

Ser Ala Gly Wall Wall Gly Met Wall Asp Ser Ile Thir Glin 825 83 O

Ala Asn Glin Luell Asp Glu Gly Pro Met Gly Glu Pro Glu Gly 84 O 845

Ser Wall Asp Glin Thir Met Wall Arg Ala Ala 855 86 O

Ile Wall Thir Wall Glin Glu Met Wall Thir ASn Thir Ser 87 O

Pro Glu Lell Gly Pro Luell Ala Asn Glin Thir Ser Asp 885 890

Gly Lell Ala Ser Glin Lys Pro Ala Ala Wall Ala Glu 905

Asn Glu Ile Gly Ala Ile His Arg Wall Glu Lell 92 O

Gly Gly Ser Ala Luell Wall Thir Ala Lell Glin 93 O 935

Pro Ser Asp Wall yr Thir Glu Lell Glu 945 950

Ala Arg Wall Ser Glu Wall Ser His Wall Lell Ala Lell 96.O 965

Glin Gly Asn Arg Gly Glin Ala Ile Ala Ser 97. 98 O

Ala Ser Gly Ile Ile Asp Luell Asp Thir Met Phe 990 995

Ala Ala Gly Thir Luell Asn Arg Glu Gly Ala Glu Thir Phe Ala 2005 2010

Asp His Arg Glu Gly Ile Luell Thir Ala Wall Luell Wall Glu 2015 2O2O 2O25

Asp Thir Wall Luell Wall Glin Asn Ala Ala Gly Ser Glin Glu 2O3O 2O35 2O4. O

Lell Ala Glin Ala Ala Glin Ser Ser Wall Ala Thir Ile Thir Arg Lell 2O45 2OSO 2O55

Ala Asp Wall Wall Luell Gly Ala Ala Ser Lell Gly Ala Glu Asp 2O60 2O65 2. Of O

Pro Thir Glin Wall Wall Luell Ile Asn Ala Wall Lys Asp Wall Ala 2O8 O 2O85

Lell Gly Asp Luell Ile Ser Ala Thir Ala Ala Ala Gly 2095 21OO

Gly Asp Asp Pro Ala Wall Trp Glin Lell Lys ASn Ser Ala 211 O 2115

Met Wall Thir Asn Wall Thir Ser Luell Lell Lys Thir Wall 2125 213 O

Ala Glu Asp Glu Ala Thir Gly Thir Arg Ala Luell Glu Ala 214 O 2145

Thir Glu His Ile Arg Glin Glu Luell Ala Wall Phe Ser Pro 215.5 216 O

Glu Pro Pro Ala Thir Ser Thir Pro Glu Asp Phe Ile Arg Met US 7,972,785 B2 111 112 - Continued

21 65 217 O 21.75

Thir Lys Gly Ile Thr Met Ala Thir Ala Ala Wall Ala Ala Gly 218O 21.85 219 O

Asn Ser Arg Glin Glu Asp Wall Ile Ala Thir Ala ASn Lell Ser 21.95 22 OO 22O5

Arg Arg Ala Ile Ala Asp Met Luell Arg Ala Lys Glu Ala Ala 221 O 2215 222 O

Phe His Pro Glu Wall Ala Pro Asp Wall Arg Lell Arg Ala Lell His 2225 223 O 2235

Phe Gly Arg Glu Cys Ala Asn Gly Tyr Luell Glu Lell Luell Asp His 224 O 2.245 225 O

Wall Luell Lell Thir Lieu. Glin Lys Pro Asn Pro Glu Lell Glin Glin 2255 226 O 2265

Lell Thir Gly His Ser Arg Wall Ala Gly Ser Wall Thir Glu Lell 2270 2275 228O

Ile Glin Ala Ala Glu Ala Met Gly Thir Glu Trp Wall Asp Pro 2285 229 O 2295

Glu Asp Pro Thir Wall Ile Ala Glu Asn Glu Lell Lell Gly Ala Ala 23 OO 23 OS 2310

Ala Ala Ile Glu Ala Ala Ala Lys Luell Glu Glin Luell Pro 2315 232O 2325

Arg Ala Pro Llys Glu Ala Asp Glu Ser Lell Asn Phe Glu Glu 233 O 2335 234 O

Glin Ile Lell Glu Ala Ala Lys Ser Ile Ala Ala Ala Thir Ser Ala 2345 2350 2355

Lell Wall Ala Ala Ser Ala Ala Glin Arg Glu Lell Wall Ala Glin 2360 23.65 2370

Gly Lys Wall Gly Ala Ile Pro Ala Asn Ala Lell Asp Asp Gly Glin 2375 238O 23.85

Trp Ser Glin Gly Lieu. Ile Ser Ala Ala Arg Met Wall Ala Ala Ala 23.90 23.95 24 OO

Thir Asn Asn Lieu. Cys Glu Ala Ala Asn Ala Ala Wall Glin Gly His 24 O5 241. O 24.15

Ala Ser Glu Lys Luell Ile Ser Ser Ala Glin Wall Ala Ala 242O 24.25 243 O

Ser Thir Gln Lieu. Luell Wall Ala Wall Lys Ala Asp Glin 2435 244 O 2445

Asp Ser Ala Met Lys Arg Luell Glin Ala Ala Gly ASn Ala Wall 2450 2455 246 O

Arg Ser Asp Asn Luell Wall Ala Ala Glin Ala Ala 24 65 2470 2475

Ala Phe Asp Glin Glu Asn Glu Thir Wall Wall Wall Glu 248O 2485 249 O

Met Wall Gly Ile Ala Glin Ile Ile Ala Ala Glin Glu Glu Met 2495 25 OO 2505

Lell Arg Glu Arg Glu Luell Glu Glu Ala Arg Lys Lell Ala 251O 25.15 252O

Glin Ile Arg Glin Glin Glin Tyr Lys Phe Luell Pro Ser Glu Lell Arg 2525 253 O 25.35

Asp Glu His 254 O

<210s, SEQ ID NO. 23 &211s LENGTH: 191 US 7,972,785 B2 113 114 - Continued

212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 23 Met Ser Ala Ala Glu Val Gly Thr Phe Val Glin Arg Lieu. Arg Gly Lieu. 1. 5 1O 15 Met Ser Glu Ile Ala Ala Phe Pro Ala Pro Thir Ile Ala Ala Met Asp 2O 25 3O Gly Phe Ala Lieu. Gly Gly Gly Lieu. Glu Lieu Ala Lieu Ala Cys Asp Lieu. 35 4 O 45 Arg Ile Ala Ala Ser Ser Ala Wal Met Gly Lieu. Ile Glu Thir Thr Arg SO 55 6 O Gly Lieu. Lieu Pro Gly Ala Gly Gly Thr Glin Arg Lieu Pro Arg Cys Lieu. 65 70 7s 8O Gly Val Ala Lieu Ala Lys Glu Lieu. Ile Phe Thr Gly Arg Arg Lieu. Asn 85 90 95 Gly Val Glin Ala His Glu Lieu. Gly Lieu Val Asn His Ala Wall Ala Glin 1OO 105 11 O Asn Glu Glu Gly Asp Ala Ala Tyr His Arg Ala Lieu Ala Lieu Ala Glin 115 12 O 125 Glu Ile Lieu Pro Glin Ala Pro Ile Ala Val Arg Lieu. Gly Llys Val Ala 13 O 135 14 O Ile Asp Arg Gly Met Glu Val Asp Ile Ala Ser Gly Met Ala Ile Glu 145 150 155 160 His Met Cys Tyr Ala Glin Asn. Ile Pro Thr Glin Asp Arg Lieu. Glu Gly 1.65 17O 17s Met Ala Ala Phe Arg Glu Lys Arg Pro Pro Llys Phe Val Gly Lys 18O 185 19 O

<210s, SEQ ID NO 24 &211s LENGTH: 425 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 24 Met Asn Tyr Ser Arg Phe Lieu. Thir Ala Thr Ser Leu Ala Arg Llys Thr 1. 5 1O 15 Ser Pro Ile Arg Ala Thr Val Glu Ile Met Ser Arg Ala Pro Lys Asp 2O 25 3O Ile Ile Ser Leu Ala Pro Gly Ser Pro Asn Pro Llys Val Phe Pro Phe 35 4 O 45 Lys Ser Ala Val Phe Thr Val Glu Asn Gly Ser Thr Ile Arg Phe Glu SO 55 6 O Gly Glu Met Phe Glin Arg Ala Leu Gln Tyr Ser Ser Ser Tyr Gly Ile 65 70 7s 8O Pro Glu Lieu. Lieu. Ser Trp Lieu Lys Glin Lieu. Glin Ile Llys Lieu. His Asn 85 90 95 Pro Pro Thr Val Asn Tyr Ser Pro Asn Glu Gly Gln Met Asp Lieu. Cys 1OO 105 11 O Ile Thr Ser Gly Cys Glin Asp Gly Lieu. Cys Llys Val Phe Glu Met Leu 115 12 O 125 Ile Asin Pro Gly Asp Thr Val Lieu Val Asn Glu Pro Leu Tyr Ser Gly 13 O 135 14 O Ala Leu Phe Ala Met Lys Pro Leu Gly Cys Asn Phe Ile Ser Val Pro 145 150 155 160

Ser Asp Asp Cys Gly Ile Ile Pro Glu Gly Lieu Lys Llys Val Lieu. Ser US 7,972,785 B2 115 116 - Continued

1.65 17O 17s

Glin Trp Pro Glu Asp Ser Asp Pro Thir Arg Thir Pro 18O 185 19 O

Phe Luell Tyr Thir Ile Pro Asn Gly Asn Asn Pro Thir Gly Asn Ser Luell 195

Thir Gly Asp Arg Glu Ile Glu Luell Ala Arg Asp 21 O 215

Phe Luell Ile Ile Glu Asp Asp Pro Phe Lell Glin Phe Thir Lys 225 23 O 235 24 O

Pro Trp Glu Pro Thir Phe Lell Ser Met Asp Wall Asp Gly Arg Wall Ile 245 250 255

Arg Ala Asp Ser Lell Ser Wall Ile Ser Ser Gly Lell Arg Wall Gly 26 O 265 27 O

Phe Ile Thir Gly Pro Ser Luell Ile Glin Arg Ile Wall Luell His Thir 27s 285

Glin Ile Ser Ser Lell His Pro Thir Luell Ser Glin Lell Met Ile Ser 29 O 295 3 OO

Glu Luell Luell Tyr Glin Trp Gly Glu Glu Gly Phe Lell Ala His Wall Asp 3. OS 310 315

Arg Ala Ile Asp Phe Asn Glin Arg Asp Phe Ile Luell Ala Ala 3.25 330 335

Ala Asp Trp Lell Arg Luell Ala Glu Trp His Wall Pro Ala 34 O 345 35. O

Gly Met Phe Luell Trp Ile Wall Asn Gly Ile Ser Asp Ala 355 360 365

Lell Ile Glu Glu Ala Ile Glu Arg Glu Ile Lell Lell Wall Pro Gly 37 O 375

Asn Ser Phe Phe Wall Asp Asn Ser Ala Pro Ser Ser Phe Phe Arg Ala 385 390 395 4 OO

Ser Phe Ser Glin Wall Thir Pro Ala Glin Met Asp Lell Wall Phe Glin Arg 4 OS 41O 415

Lell Ala Glin Luell Ile Asp Wall Ser 425

SEO ID NO 25 LENGTH: 699 TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 25

Met Glu Wal His Glu Lell Phe Arg Phe Arg Met Pro Glu Luell Ile 1. 5 15

Asp Ile Arg Glin Tyr Wall Arg Thir Luell Pro Thir Asn Thir Luell Met Gly 2O 25

Phe Gly Ala Phe Ala Ala Lell Thir Thir Phe Trp Ala Thir Arg Pro 35 4 O 45

Ala Luell Pro Pro Cys Asp Luell Ser Met Glin Ser Wall Glu Wall SO 55 6 O

Gly Thir Thir Glu Gly Wall Arg Arg Ser Ala Wall Lell Glu Asp Asp 70

Luell Luell Luell Tyr Asp Asp Wall Arg Thir Met Asp Gly 85 90 95

Phe Glin Arg Gly Ile Glin Wall Ser Asn Asp Gly Pro Luell Gly Ser 1OO 105 11 O

Arg Pro Asn Glin Pro Glu Trp Ile Ser Glin Wall Ala US 7,972,785 B2 117 118 - Continued

115 12 O 125

Glu Met Ala Glu Cys Ile Ser Ala Luell Ile Glin Lys Gly Phe Lys 13 O 14 O

Pro Ser Glu Glin Phe Gly Ile Phe Ser Glin Asn Arg Pro Glu 145 150 155 160

Trp Wall Thir Ile Glu Glin Phe Thir Ser Met Wall Wall Wall 1.65 17s

Pro Luell Asp Thir Lell Thir Asp Ala Ile Thir Ile Wall Asn 18O 185 19 O

Ala Glu Luell Ser Wall Phe Ala Asp Pro Glu Ala Lys 195

Lell Luell Luell Glu Gly Wall Asn Luell Thir Pro Luell Ile 21 O 22O

Ile Wall Ile Met Asp Ser Asp Asn Asp Luell Wall Glu Arg Gly Glin 225 23 O 235 24 O

Gly Wall Glu Ile Ile Gly Luell Lys Ala Lell Glu Asp Luell Gly 245 250 255

Arg Wall Asn Arg Thir Pro Pro Pro Glu Pro Glu Asp Luell Ala 26 O 265 27 O

Ile Ile Cys Phe Thir Ser Gly Thir Thir Gly ASn Pro Lys Gly Ala Met 27s 28O 285

Wall Thir His Glin Asn Ile Met Asn Asp Ser Gly Phe Ile Ala 29 O 295 3 OO

Thir Glu Ser Ala Phe Ile Ala Ser Pro Glu Asp Wall Lell Ile Ser Phe 3. OS 310 315

Lell Pro Luell Ala His Met Phe Glu Thir Wall Wall Glu Wall Met Luell 3.25 330 335

His Gly Ala Lys Ile Gly Phe Phe Glin Gly Asp Ile Arg Luell Luell 34 O 345 35. O

Met Asp Asp Luell Lys Wall Lell Glin Pro Thir Ile Phe Pro Wall Wall Pro 355 360 365

Arg Luell Luell Asn Arg Met Phe Asp Arg Ile Phe Gly Glin Ala Asn Thir 37 O 375

Ser Wall Arg Trp Lell Lell Asp Phe Ala Ser Arg Glu Ala 385 390 395 4 OO

Glu Luell Arg Ser Gly Ile Wall Arg Asn Asn Ser Lell Trp Asp Lys Luell 4 OS 415

Ile Phe His Lys Ile Glin Ser Ser Luell Gly Gly Wall Arg Luell Met 425 43 O

Ile Thir Gly Ala Ala Pro Wall Ser Ala Thir Wall Lell Thir Phe Luell Arg 435 44 O 445

Ala Ala Luell Gly Cys Glin Phe Glu Gly Gly Glin Thir Glu Cys 450 45.5 460

Thir Ala Gly Cys Lell Ser Luell Pro Gly Asp Trp Thir Ala Gly His 465 470

Wall Gly Ala Pro Met Pro Asn Ile Lell Wall Asp Wall Glu 485 490 495

Asp Met Asn Tyr Glin Ala Ala Gly Glu Gly Glu Wall Cys Wall Lys SOO 505

Gly Ala Asn Wall Phe Gly Tyr Luell Lys Asp Pro Ala Arg Thir Ala 515 52O 525

Glu Ala Luell Asp Lys Asp Gly Trp Luell His Thir Gly Asp Ile Gly Lys 53 O 535 54 O US 7,972,785 B2 119 120 - Continued

Trp Luell Pro Asn Gly Thir Lell Ile Ile Asp Arg Lys Lys His Ile 5.45 550 555 560

Phe Luell Ala Glin Gly Ile Ala Pro Glu Lys Ile Glu Asn 565 st O sts

Ile Luell Arg Ser Glu Wall Ala Glin Wall Phe Wall His Gly Glu 585 59 O

Ser Luell Glin Ala Phe Lell Ala Ile Wall Wall Pro Asp Wall Glu Ile 595 605

Lell Pro Ser Trp Ala Glin Arg Gly Phe Glin Gly Ser Phe Glu Glu 610

Lell Arg Asn Asp Asn Ala Ile Lell Glu Asp Met Wall 625 630 635 64 O

Luell Gly Asn Ala Luell Pro Phe Glu Glin Wall Lys Gly 645 650 655

Ile Ala Wall His Pro Glu Lell Phe Ser Ile Asp Asn Gly Luell Luell Thir 660 665 67 O

Pro Thir Luell Ala Pro Glu Luell Arg Asn Tyr Phe Arg Ser 675 685

Glin Ile Asp Glu Lell Ser Thir Ile Ile 69 O. 695

SEQ ID NO 26 LENGTH: 902 TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 26

Met Lys Ile Ala Ile Gly Glin Ser Luell Phe Gly Glin Glu Wall 1. 15

Cys Glin Luell Arg Glu Gly His Glu Wall Wall Gly Wall Phe Thir Ile 25

Pro Asp Lys Asp Gly Ala Asp Pro Luell Gly Lell Glu Ala Glu 35 4 O 45

Asp Gly Arg Ala Wall Phe Lys Phe Pro Arg Trp Arg Ala Arg Gly Glin SO 55 6 O

Ala Luell Pro Glu Wall Wall Ala Glin Ala Lell Gly Ala Glu Luell 65 70 8O

Asn Wall Luell Pro Phe Ser Glin Phe Ile Pro Met Glu Wall Ile Asn 85 90 95

Ala Pro Arg His Gly Ser Ile Ile Tyr His Pro Ser Lell Luell Pro Arg 105 11 O

His Arg Gly Ala Ser Ala Ile Asn Trp Thir Luell Ile His Gly Asp 115 12 O 125

Gly Gly Phe Thir Ile Phe Trp Ala Asp Asp Gly Lell Asp Thir Gly 13 O 135 14 O

Asp Luell Luell Luell Glin Lys Glu Glu Wall Luell Pro Asp Asp Thir Wall 145 150 155 160

Ser Thir Luell Tyr Asn Arg Phe Luell Phe Pro Glu Gly Ile Gly Met 1.65 17O 17s

Wall Glin Ala Wall Arg Lell Ile Ala Glu Gly Thir Ala Pro Arg Pro 18O 185 19 O

Glin Ser Glu Glu Gly Ala Thir Tyr Glu Gly Ile Glin Lys Glu Thir 195 2OO

Ala Lys Ile Asn Trp Asp Glin Pro Ala Glu Ala Ile His Asn Trp Ile 21 O 215 22O US 7,972,785 B2 121 122 - Continued

Arg Gly Asn Asp Wall Pro Gly Ala Trp Thir Glu Ala Cys Gly Glin 225 23 O 235 24 O

Luell Thir Phe Phe Asn Ser Thir Luell Asn Thir Ser Gly Luell Ser Thir 245 250 255

Glin Gly Glu Lell Pro Ile Pro Gly Ala His Arg Pro Gly Wall Wall 265 27 O

Thir Ala Lell Ile Lell Phe Gly Asn Glu His Arg Met Luell Luell 28O 285

Wall Lys Asn Glin Lell Glu Asp Gly Met Met Pro Ala Ser Glin 29 O 295 3 OO

Phe Phe Ser Ala Ser Ser Asp Luell Glu Lell Thir Glu Ala Glu 3. OS 310 315

Lell Ala Thir Glu Ala Wall Arg Ser Ser Trp Met Arg Ile Luell Pro 3.25 330 335

Asn Wall Pro Wall Glu Asp Ser Thir Asp Phe Phe Ser Gly Ala 345 35. O

Ala Ser Wall Asp Wall Wall Arg Luell Wall Glu Glu Wall Lys Glu Luell 355 360 365

Asp Gly Luell Glu Lell Glu Asn Glu Asp Wall Met Ala Thir Thir Phe 37 O 375

Arg Glu Phe Ile Glin Lell Lell Wall Arg Luell Arg Gly Glu Asp Asp 385 390 395 4 OO

Glu Ser Glu Wall Ile Asn Wall Glu Arg Ala Wall Asn Lys Luell 4 OS 415

Thir Luell Glin Met Pro Glin Luell Phe Ile Gly Gly Glu Phe Wall Asp 425 43 O

Ala Glu Gly Ser Thir Asn Thir Ile ASn Pro Thir Asp Gly Ser 435 44 O 445

Wall Ile Glin Wall Ser Lell Ala Glin Wall Ser Asp Wall Asp Ala 450 45.5 460

Wall Ala Ala Ala Ala Phe Glu Asn Gly Lell Trp Gly Ile 465

Asn Ala Arg Asp Arg Arg Luell Luell Tyr Arg Lell Ala Asp Wall Met 485 490 495

Glu Glin His Glin Glu Lell Ala Thir Ile Glu Ala Lell Asp Arg Gly SOO 505

Ala Wall Tyr Thir Lell Lell Lys Thir His Wall Gly Met Ser Ile Glin 515 525

Thir Phe Arg Phe Gly Trp Asp Ile Glin Gly Ala Thir 53 O 535 54 O

Ile Pro Ile Asn Glin Arg Pro Asn Arg ASn Lell Thir Luell Thir Lys 5.45 555 560

Glu Pro Wall Gly Gly Ile Wall Ile Pro Trp Asn Tyr Pro 565 st O sts

Lell Met Met Luell Ser Trp Thir Ala Ala Lell Ala Ala Gly Asn 585 59 O

Thir Wall Wall Ile Pro Ala Glin Wall Thir Pro Lell Thir Ala Luell 595 605

Phe Ala Glu Luell Thir Lell Lys Ala Gly Ile Pro Lys Gly Wall Wall Asn 610 615

Ile Luell Pro Gly Ser Gly Ser Luell Wall Gly Glin Arg Lell Ser Asp His 625 630 635 64 O

Pro Asp Wall Arg Lys Ile Gly Phe Thir Gly Ser Thir Glu Wall Gly Lys 645 650 655 US 7,972,785 B2 123 124 - Continued

His Ile Met Lys Ser Ala Luell Ser Asn Wall Lys Lys Wall Ser Luell 660 665 67 O

Glu Luell Gly Gly Lys Ser Pro Luell Ile Ile Phe Ala Asp Asp Luell 675 685

Asn Lys Ala Wall Glin Met Gly Met Ser Ser Wall Phe Phe Asn Gly 69 O. 695 7 OO

Glu Asn Ile Ala Ala Gly Arg Luell Phe Wall Glu Glu Ser Ile His 7 Os

Asn Glin Phe Wall Glin Wall Wall Glu Glu Wall Glu Met Lys Ile 72 73 O 73

Gly Asn Pro Luell Glu Arg Asp Thir Asn His Gly Pro Glin Asn His Glu 740 74. 7 O

Ala His Luell Arg Lys Lell Wall Glu Glin Arg Gly Wall Glu 760 765

Gly Ala Thir Luell Wall Gly Gly Asn Glin Wall Pro Arg Pro Phe 770 775

Phe Phe Glin Pro Thir Wall Phe Thir Asp Wall Glu Asp His Met Ile 79 O 79.

Ala Glu Glu Ser Phe Gly Pro Ile Met Ile Ile Ser Arg Phe Ala 805 810 815

Asp Gly Asp Wall Asp Ala Wall Luell Ser Arg Ala Asn Ala Thir Glu Phe 825 83 O

Gly Luell Ala Ser Gly Wall Phe Thir Arg Asp Ile Asn Lys Ala Luell 835 84 O 845

Wall Ser Asp Lell Glin Ala Gly Thir Wall Phe Ile Asn Thir Asn 850 855 860

Lys Thir Asp Wall Ala Ala Pro Phe Gly Gly Phe Glin Ser Gly Phe 865 88O

Gly Asp Luell Gly Glu Ala Ala Luell Asn Glu Lell Arg Ile 885 890 895

Thir Wall Thir Phe Glu 9 OO

<210s, SEQ ID NO 27 &211s LENGTH: 412 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 27

Met Ser Ser Lys Gly Ser Wall Wall Luell Ala Ser Gly Gly Luell Asp 1. 5 1O 15

Thir Ser Cys Ile Lell Wall Trp Luell Lys Glu Glin Gly Asp Wall Ile 2O 25

Ala Luell Ala Asn Ile Gly Glin Glu Asp Phe Glu Glu Ala Arg 35 4 O 45

Ala Luell Lys Lell Gly Ala Wall Phe Ile Glu Asp Wall 55 6 O

Ser Glu Phe Wall Glu Glu Phe Ile Trp Pro Ala Wall Glin Ser Ser 65 70

Ala Luell Glu Asp Arg Luell Luell Gly Thir Ser Lell Ala Arg Pro 85 90 95

Ile Ala Arg Lys Glin Wall Glu Ile Ala Glin Arg Glu Gly Ala 105 11 O

Wall Ser His Gly Ala Thir Gly Gly ASn Asp Glin Wall Arg Phe 115 12 O 125 US 7,972,785 B2 125 126 - Continued

Glu Luell Thir Tyr Ser Lell Ala Pro Glin Ile Lys Wall Ile Ala Pro 13 O 135 14 O

Trp Arg Met Pro Glu Phe Asn Arg Phe Lys Gly Arg Asn Asp Luell 145 150 155 160

Met Glu Tyr Ala Lys Glin His Gly Ile Pro Ile Pro Wall Thir Pro 1.65 17O 17s

Ser Pro Trp Ser Met Asp Glu Asn Luell Met His Ile Ser Tyr Glu Ala 18O 185 19 O

Gly Ile Luell Glu Asn Pro Asn Glin Ala Pro Pro Gly Luell Tyr Thir 195

Thir Glin Asp Pro Ala Lys Ala Pro Asn Thir Pro Asp Wall Luell Glu 21 O 215 22O

Ile Glu Phe Lys Gly Wall Pro Wall Wall Thir Asn Wall Asp 225 23 O 235 24 O

Gly Thir Thir His Ser Thir Ser Luell Asp Luell Phe Met Luell Asn Glu 245 250 255

Wall Ala Gly Lys His Gly Wall Gly Arg Ile Asp Ile Wall Glu Asn Arg 26 O 265 27 O

Phe Ile Gly Met Lys Ser Arg Gly Ile Glu Thir Pro Ala Gly Thir 285

Ile Luell Tyr His Ala His Lell Asp Ile Glu Ala Phe Thir Met Asp Arg 29 O 295 3 OO

Glu Wall Arg Ile Lys Glin Gly Luell Gly Luell Lys Phe Ala Glu Luell 3. OS 310 315

Wall Thir Gly Phe Trp His Ser Pro Glu Cys Glu Phe Wall Arg His 3.25 330 335

Ile Asp Lys Ser Glin Glu Arg Wall Glu Gly Lys Wall Glin Wall Ser 34 O 345 35. O

Wall Phe Lys Gly Glin Wall Ile Luell Gly Arg Glu Ser Pro Luell Ser 355 360 365

Lell Tyr Asn Glu Glu Lell Wall Ser Met Asn Wall Glin Gly Asp Glu 37 O 375

Pro Ile Asp Ala Thir Gly Phe Ile Asn Ile ASn Ser Lell Arg Luell Lys 385 390 395 4 OO

Glu His Arg Lell Glin Ser Wall Thir Ala 4 OS 41O

<210s, SEQ ID NO 28 &211s LENGTH: 857 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 28

Met Lieu. Arg Lieu Gly Ala Lell Arg Luell Arg Gly Lell Ala Luell Arg Ser 1. 5 1O 15

Ser Glin Gly Arg Pro Ser Ser Ala Gly Luell Arg Glu Gly Glin Glu Ser 2O 25

Pro Pro Ser Pro Pro Glu Trp Lys Asp Arg Ala Glu Thir Wall Ile Ile 35 4 O 45

Gly Gly Gly Wall Gly Wall Ser Luell Ala His Lell Ala Ala SO 55 6 O

Gly Met Arg Asp Wall Wall Lell Luell Glu Glu Lell Thir Ala Gly 65 70

Ser Thir Trp His Ala Ala Gly Luell Thir Thir Phe His Pro Gly Ile 85 90 95 US 7,972,785 B2 127 128 - Continued

Asn Luell Lys Ile His Asp Ser Ile Lys Lell Tyr Glu Arg Luell 105 11 O

Glu Glu Glu Thir Gly Glin Wall Wall Gly Phe His Glin Pro Gly Ser Ile 115 12 O 125

Arg Luell Ala Thir Thir Pro Glu Arg Wall Asp Glu Phe Glin Met 13 O 135 14 O

Thir Arg Thir Asn Trp His Ala Thir Glu Glin Tyr Ile Ile Glu Pro Glu 145 150 155 160

Ile His Glu Lell Phe Pro Luell Luell Asn Met Asp Ile Luell Ala 1.65 17O 17s

Gly Luell Asn Pro Gly Asp Gly His Ile Asp Pro Ser Luell Thir 18O 185 19 O

Met Ala Luell Ala Thir Gly Ala Arg Gly Ala Lell Luell 195

Pro Ala Pro Wall Thir Ser Lell Pro Arg Pro Asp Gly Thir Trp Asp 21 O 215

Wall Glu Thir Pro Glin Gly Ser Wall Arg Ala ASn Arg Ile Wall Asn Ala 225 23 O 235 24 O

Ala Gly Phe Trp Ala Arg Glu Wall Gly Lys Met Ile Gly Luell Asp His 245 250 255

Pro Luell Ile Pro Wall Glin His Glin Tyr Wall Ile Thir Ser Thir Ile Pro 26 O 265 27 O

Glu Wall Lys Ala Lell Arg Glu Luell Pro Wall Lell Arg Asp Luell Glu 27s 285

Gly Ser Lell Arg Glin Glu Arg Asp Gly Lell Lell Phe Gly Pro 29 O 295 3 OO

Tyr Glu Ser Glin Glu Lys Met Luell Glin Ala Ser Trp Wall Ala His 3. OS 310 315

Gly Wall Pro Pro Gly Phe Gly Glu Luell Phe Glu Ser Asp Luell Asp 3.25 330 335

Arg Ile Thir Glu His Wall Glu Ala Ala Met Glu Met Wall Pro Wall Luell 34 O 345 35. O

Ala Asp Ile Ile Asn Ile Wall Asn Gly Pro Ile Thir Ser 355 360 365

Pro Asp Ile Luell Pro Met Wall Gly Pro His Glin Gly Wall Arg Asn 37 O 375

Trp Wall Ala Ile Gly Phe Gly Gly Ile Ile His Ala Gly Gly Wall 385 390 395 4 OO

Gly Thir Luell Ser Asp Trp Ile Luell His Gly Glu Pro Pro Phe Asp 4 OS 415

Lell Ile Glu Luell Asp Pro Asn Arg Tyr Gly Trp Thir Thir Thir Glin 425 43 O

Thir Glu Ala Lys Ala Arg Glu Ser Gly Phe Asn Asn Ile Wall 435 44 O 445

Gly Tyr Pro Glu Glu Arg Phe Ala Arg Pro Thir Glin Arg Wall 450 45.5 460

Ser Gly Luell Lys Ile Lell Glu Ser Cys Ser Met Gly Phe His 465 470

Ala Gly Trp Glu Glin Pro His Trp Phe Tyr Pro Gly Glin Asp Thir 485 490 495

Glin Arg Pro Ser Phe Arg Arg Thir Asn Trp Phe Glu Pro Wall Gly SOO 505 51O

Ser Glu Glin Wall Met Glin Arg Wall Gly Wall Ile Asp Luell Ser US 7,972,785 B2 129 130 - Continued

515 525

Pro Phe Gly Lys Phe Asn Ile Gly Glin Asp Ser Thir Glin Luell Luell 53 O 535 54 O

Asp His Luell Cys Ala Asn Wall Ile Pro Wall Gly Phe Thir Asn Ile 5.45 550 555 560

Ser His Met Luell Thir Pro Arg Gly Arg Wall Tyr Ala Glu Luell Thir Wall 565 st O sts

Ser His Glin Ser Pro Gly Glu Phe Luell Luell Thir Gly Ser Gly Ser 585 59 O

Glu Luell His Asp Lell Arg Trp Ile Glu Glu Ala Wall Arg Gly Gly 595 605

Asp Wall Glu Ile Arg Asn Ile Thir Asp Lell Gly Wall Luell Gly 610 615

Wall Ala Gly Pro Tyr Ala Arg Arg Wall Luell Lys Lell Thir Ser Glu 625 630 64 O

Asp Luell Ser Asp Asp Wall Phe Phe Luell Thir Ser Luell 645 650 655

Ile Ser Asp Ile Pro Wall Thir Ala Ile Arg Ser Thir Gly Glu 660 665 67 O

Lell Gly Trp Glu Lell Tyr His Arg Arg Glu Asp Ser Ala Ala Luell Tyr 675 685

Glu Arg Met Asn Ala Gly Glin Glu Glu Gly Ile Asp Asn Phe Gly 69 O. 695 7 OO

Thir Luell Asn Ala Lell Arg Luell Glu Lys Ala Phe Arg Ala Trp 7 Os

Gly Ser Met Asn Asp Thir Asn Pro Luell Glu Ala Gly Luell Asp 72 73 O 73

Phe Lys Lell Asn Pro Ala Asn Phe Thir Gly Lys Glin Ala 740 74. 7 O

Lell Ile Ala Gly Luell Arg Arg Lell Wall Luell 760 765

Thir Thir Asp Asn Wall Asp Pro Glu Gly Asn Glu Ser Wall Trp 770 775

Tyr Wall Ile Gly Asn Thir Thir Ser Gly Ser Ser Tyr 79 O 79.

Ser Ile Ser Lell Ala Phe Ala Tyr Wall Pro Wall Glu Luell Ser 805 810 815

Glu Wall Glin Glin Wall Glu Wall Glu Luell Luell Gly Asn Tyr Pro 82O 825 83 O

Ala Thir Ile Glin Glu Pro Luell Wall Luell Thir Glu Pro Thir Arg Thir 84 O 845

Arg Asp Gly Arg Ser 850 855

<210s, SEQ ID NO 29 &211s LENGTH: 519 212. TYPE : PRT <213> ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 29 Met Ala Pro Glin Glu Arg Lieu. Lieu. Ile Arg Gly Gly Arg Wal Wall Asn 1. 5 15 Asp Asp Phe Ser Glin Val Ala Asp Val Lieu Val Glu Asp Gly Val Val 25

Arg Ala Lieu. Gly Arg Asp Lieu. Lieu Pro Pro Gly Asp Thr Ser Arg Gly US 7,972,785 B2 131 132 - Continued

35 4 O 45

Lell Arg Ile Luell Asp Ala Ala Gly Luell Wall Lell Pro Gly Gly Ile SO 55 6 O

Asp Thir His Thir His Met Glin Phe Pro Phe Met Gly Ser Glin Ser Wall 65 70 8O

Asp Asp Phe His Glin Gly Thir Ala Ala Luell Ala Gly Gly Th Thr 85 90 95

Met Ile Ile Asp Phe Ala Ile Pro Glin Gly Ser Ser Luell Ile Glu 105 11 O

Ala Phe Glu Thir Trp Arg Asn Trp Ala Asp Pro Wall 115 12 O 125

Ser Luell His Wall Ala Wall Thir Trp Trp Ser Asp Wall Lys Glu 13 O 135 14 O

Glu Met Thir Lell Ala Glin Asp Gly Wall Asn Ser Phe Lys Met 145 150 155 160

Phe Met Ala Lys Asp Lell Tyr Met Wall Glin Asp Glin Glin Met Tyr 1.65 17O 17s

Ala Ala Phe Ser Glin Glu Ile Gly Ala Ile Ala Glin Wall His 18O 185 19 O

Ala Glu Asn Gly Asp Lell Ile Ala Glu Gly Ala Lys Met Lieu Ala 195

Lell Gly Ile Thir Gly Pro Glu Gly His Glu Luell Cys Arg Pro Glu Ala 21 O 215 22O

Wall Glu Ala Glu Ala Thir Lell Arg Ala Ile Thir Ile Ala Ser Ala Wall 225 23 O 235 24 O

Asn Pro Luell Tyr Ile Wall His Wall Met Ser Ser Ala Ala Lys 245 250 255

Wall Ile Ala Asp Ala Arg Glu Gly Lys Wall Wall Gly Glu Pro 26 O 265 27 O

Ile Ala Ala Gly Lell Gly Thir Asp Gly Thir Glin Tyr Trp Asn Lys Glu 285

Trp Arg His Ala Ala His His Wall Met Gly Pro Pro Lell Arg Pro Asp 29 O 295 3 OO

Pro Ser Thir Pro Gly Phe Lell Met Asn Luell Luell Ala Asn Gly Asp Lieu. 3. OS 310 315 32O

Thir Thir Thir Gly Ser Asp Asn Thir Phe ASn Thir Glin Lys Ala 3.25 330 335

Lell Gly Asp Asp Phe Thir Ile Pro ASn Gly Wall Asn Gly Val 34 O 345 35. O

Glu Asp Arg Met Ser Wall Ile Trp Glu Gly Wall His Ser Gly Lys 355 360 365

Met Asp Glu Asn Arg Phe Wall Ala Wall Thir Ser Thir Asn Ala Ala Lys 37 O 375

Ile Phe Asn Luell Tyr Pro Gly Arg Ile Ala Wall Gly Ser Asp 385 390 395 4 OO

Ala Asp Met Wall Ile Trp Asp Pro Glu Ala Thir Arg Thir Ile Ser Ala 4 OS 415

Thir His His Glin Ala Wall Asn Phe Asn Ile Phe Glu Gly Met Wall 425 43 O

His Gly Wall Pro Lell Wall Thir Ile Ser Arg Gly Arg Wall Val Tyr 435 44 O 445

Glu Ala Gly Wall Phe Asp Wall Thir Ala Gly His Gly Lys Phe Ile Pro 450 45.5 460 US 7,972,785 B2 133 134 - Continued

Arg Glin Pro Phe Ala Glu Phe Ile Tyr Arg Val Lys Glin Arg Asp 465 470 47s 48O

Glin Thir Thir Pro Ile Pro Wall Lys Arg Ala Pro Tyr Lys Gly Glu 485 490 495

Wall Ile Thir Lieu Lys Pro Arg Glu Thir Glu Asp Asp Thir Ala Gly SOO 505

Thir Arg Met Gln Gly His Ser 515

<210s, SEQ ID NO 3 O &211s LENGTH: 885 212. TYPE : PRT &213s ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 30

Met Ala Arg Glin Gly Lell Gly Ser Phe Glin Wall Ile Ser Luell Phe 1. 5 1O 15

Thir Phe Ala Ile Ser Wall Asn Ile Cys Luell Gly Phe Thir Ala Ser Arg 2O 25 3O

Ile Arg Ala Glu Trp Asp Glu Gly Pro Pro Thir Wall Luell Ser Asp 35 4 O 45

Ser Pro Trp Thir Asn Thir Ser Gly Ser Gly Arg Phe Glu SO 55 6 O

Lell Glin Glu Val Gly Pro Pro Asp Arg Cys Asp Asn Luell Lys 65 70 7s

Ser Ser Ser Cys His Asp Phe Asp Glu Lell Luell Lys Thir 85 90 95

Wall Arg Gly Trp. Glu Thir Asp Arg Ser Gly Glu Wall Arg Asn 1OO 105 11 O

Glu Glu Asn Ala Cys His Pro Glu Asp Lell Ser Arg Gly Asp 115 12 O 125

Cys Thir Asn Tyr Glin Wall Wall Cys Gly Glu Ser His Trp Wall 13 O 135 14 O

Asp Asp Ala Ala Arg Asn Glin Ser Ser Glu Cys Lell Glin Wall Pro 145 150 155 160

Pro Pro Luell Ile Ile Phe Ser Wall Asp Gly Phe Arg Ala Ser Tyr Met 1.65 17O 17s

Gly Ser Lys Wall Met Pro Asn Ile Glu Lell Arg Ser 18O 185 19 O

Gly Thir His Wall Pro Thir Arg Pro Wall Pro Thir Thir Phe 195

Pro Asn Luell Tyr Thr Lell Ala Thir Gly Luell Tyr Pro Glu Ser His Gly 21 O 215 22O

Ile Wall Gly Asn. Ser Met Asp Pro Wall Phe Asp Ala Ser Phe His 225 23 O 235 24 O

Lell Arg Gly Arg Glu Phe Asn His Arg Trp Trp Gly Gly Glin Pro 245 250 255

Lell Trp Ile Thir Ala Thir Glin Gly Wall Arg Ala Gly Thir Phe Phe 26 O 265 27 O

Trp Ser Wall Ser Ile Pro His Glu Arg Arg Ile Lell Thir Ile Luell Glin 285

Trp Luell Ser Leul Pro Asp Asn Glu Arg Pro Ser Wall Ala Phe Tyr 29 O 295 3 OO

Ser Glu Glin Pro Asp Phe Ser Gly His Tyr Gly Pro Phe Gly Pro 3. OS 310 315 32O US 7,972,785 B2 135 136 - Continued

Glu Met Thir Asn Pro Lell Arg Glu Ile Asp Thir Wall Gly Glin Luell 3.25 330 335

Met Asp Gly Luell Glin Lell Arg Luell His Arg Wall Asn Wall Ile 34 O 345 35. O

Phe Wall Gly Asp His Gly Met Glu Asp Wall Thir Asp Arg Thir Glu 355 360 365

Phe Luell Ser Asn Tyr Lell Thir Asn Wall Asp Asp Ile Thir Luell Wall Pro 37 O 375 38O

Gly Thir Luell Gly Arg Ile Arg Ala Ser Ile Asn Asn Ser Tyr 385 390 395 4 OO

Asp Pro Thir Ile Ile Ala Asn Luell Thir Pro Asp Glin 4 OS 415

His Phe Pro Tyr Met Glin His Luell Pro Arg Luell His Tyr 425 43 O

Ala Asn Asn Arg Arg Ile Glu Asp Ile His Luell Lell Wall Asp Arg Arg 435 44 O 445

Trp His Wall Ala Arg Pro Luell Asp Wall Lys Pro Ser Gly 450 45.5 460

Lys Phe Phe Glin Gly Asp His Gly Phe Asp Asn Wall Asn Ser 465 470

Met Glin Thir Wall Phe Wall Gly Tyr Gly Pro Thir Phe Arg Thir 485 490 495

Wall Pro Pro Phe Glu Asn Ile Glu Luell Tyr Asn Wall Met Asp SOO 505

Lell Luell Gly Luell Pro Ala Pro Asn Asn Gly Thir His Gly Ser Luell 515 525

Asn His Luell Luell Arg Thir Asn Thir Phe Arg Pro Thir Met Pro Asp Glu 53 O 535 54 O

Wall Ser Arg Pro Asn Tyr Pro Gly Ile Met Tyr Lell Glin Ser Glu Phe 5.45 550 555 560

Asp Luell Gly Thir Asp Asp Wall Glu Pro Asn Lys Luell 565 st O sts

Glu Glu Luell Asn Arg Lell His Thir Gly Ser Thir Glu Ala Glu 585 59 O

Thir Gly Lys Phe Gly Ser Lys His Glu ASn Lys Asn Luell Asn 595 605

Gly Ser Wall Glu Pro Arg Lys Glu Arg His Luell Lell Tyr Gly Arg Pro 610 615

Ala Wall Luell Tyr Thir Ser Asp Ile Luell Tyr His Thir Asp Phe 625 630 635 64 O

Glu Ser Gly Tyr Ser Glu Ile Phe Luell Met Pro Lell Trp Thir Ser Tyr 645 650 655

Thir Ile Ser Lys Glin Ala Glu Wall Ser Ser Ile Pro Glu His Luell Thir 660 665 67 O

Asn Wall Arg Pro Asp Wall Arg Wall Ser Pro Gly Phe Ser Glin Asn 675 68O 685

Luell Ala Lys Asn Asp Glin Met Ser Tyr Gly Phe Luell Phe 69 O. 695 7 OO

Pro Pro Luell Ser Ser Ser Pro Glu Ala Lys Asp Ala Phe Luell 7 Os

Wall Thir Asn Met Wall Pro Met Pro Ala Phe Arg Wall Trp Ala 72 73 O 73

Phe Glin Arg Wall Lell Wall Lys Ala Ser Glu Arg Asn Gly 740 74. 7 O US 7,972,785 B2 137 138 - Continued

Wall Asn Wall Ile Ser Gly Pro Ile Phe Asp Asn Tyr Asp Gly Luell 760 765

Arg Asp Thir Glu Asp Glu Ile Glin Tyr Wall Glu Gly Ser Ser Ile 770 775 78O

Pro Wall Pro Thir His Tyr Ser Ile Ile Thir Ser Cys Luell Asp Phe 79 O 79.

Thir Glin Pro Ala Asp Asp Gly Pro Luell Ser Wall Ser Ser Phe 805 810 815

Ile Luell Pro His Arg Pro Asp Asn Asp Glu Ser Asn Ser Ser Glu 825 83 O

Asp Glu Ser Trp Wall Glu Glu Luell Met Met His Thir Ala Arg 835 84 O 845

Wall Arg Asp Ile Glu His Lell Thir Gly Luell Asp Phe Arg Thir 850 855 860

Ser Arg Ser Ser Glu Ile Luell Thir Luell Lys Thir Luell His Thir 865 87O 87s 88O

Glu Ser Glu Ile 885

<210s, SEQ ID NO 31 &211s LENGTH: st 9 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 31

Met Wall Leu Ser Glu Wall Trp Thir Thir Luell Ser Gly Wall Ser Gly Wall 1. 5 15

Cys Luell Ala Cys Ser Lell Lell Ser Ala Ala Wall Wall Lell Arg Trp Thir 25

Gly Arg Glin Lys Ala Arg Gly Ala Ala Thir Arg Ala Arg Glin Glin 35 4 O 45

Arg Ala Ser Luell Glu Thir Met Asp Ala Wall Glin Arg Phe Arg Luell SO 55 6 O

Glin Asn Pro Asp Lell Asp Ser Glu Ala Luell Luell Thir Lell Pro Luell Luell 65 70

Glin Luell Wall Glin Lys Lell Glin Ser Gly Glu Luell Ser Pro Glu Ala Wall 85 90 95

Phe Phe Thir Tyr Lell Gly Ala Trp Glu Wall Asn Gly Thir Asn 105 11 O

Wall Thir Ser Tyr Lell Thir Asp Glu Thir Glin Lell Ser Glin Ala 115 12 O 125

Pro Arg Glin Gly Lell Lell Tyr Gly Wall Pro Wall Ser Lell Glu 13 O 135 14 O

Phe Ser Gly His Asp Ser Thir Luell Gly Lell Ser Luell Asn Glu 145 150 155 160

Gly Met Pro Ser Glu Ser Asp Wall Wall Wall Glin Wall Luell Lys Luell 1.65 17O 17s

Glin Gly Ala Wall Pro Phe Wall His Thir Asn Wall Pro Glin Ser Met Luell 18O 185 19 O

Ser Phe Asp Ser Asn Pro Luell Phe Gly Glin Thir Met Asn Pro Trp 195 2OO

Ser Ser Ser Pro Gly Gly Ser Ser Gly Gly Glu Gly Ala Luell 21 O 215 22O

Ile Gly Ser Gly Gly Ser Pro Luell Gly Luell Gly Thir Asp Ile Gly Gly 225 23 O 235 24 O US 7,972,785 B2 139 140 - Continued

Ser Ile Arg Phe Pro Ser Ala Phe Gly Ile Cys Gly Luell Lys Pro 245 250 255

Thir Gly Asn Arg Lell Ser Ser Gly Luell Gly Cys Wall Tyr Gly 26 O 265 27 O

Glin Thir Ala Wall Glin Lell Ser Luell Gly Pro Met Ala Arg Asp Wall Glu 285

Ser Luell Ala Luell Cys Lell Lys Ala Luell Luell Glu His Luell Phe Thir 29 O 295 3 OO

Lell Asp Pro Thir Wall Pro Pro Luell Pro Phe Arg Glu Glu Wall Arg 3. OS 310 315

Ser Ser Arg Pro Lell Arg Wall Gly Tyr Glu Thir Asp Asn Tyr Thir 3.25 330 335

Met Pro Ser Pro Ala Met Arg Arg Ala Luell Ile Glu Thir Lys Glin Arg 34 O 345 35. O

Lell Glu Ala Ala Gly His Thir Luell Ile Pro Phe Lell Pro Asn Asn Ile 355 360 365

Pro Tyr Ala Luell Glu Wall Lell Ser Ala Gly Gly Lell Phe Ser Asp Gly 37 O 375

Gly Arg Ser Phe Lell Glin Asn Phe Gly Asp Phe Wall Asp Pro Cys 385 390 395 4 OO

Lell Gly Asp Luell Ile Lell Ile Luell Arg Luell Pro Ser Trp Phe Lys Arg 4 OS 415

Lell Luell Ser Luell Lell Lell Pro Luell Phe Pro Arg Lell Ala Ala Phe 425 43 O

Lell Asn Ser Met Arg Pro Arg Ser Ala Glu Lys Lell Trp Luell Glin 435 44 O 445

His Glu Ile Glu Met Arg Glin Ser Wall Ile Ala Glin Trp Ala 450 45.5 460

Met Asn Luell Asp Wall Lell Lell Thir Pro Met Luell Gly Pro Ala Luell Asp 465 470

Lell Asn Thir Pro Gly Arg Ala Thir Gly Ala Ile Ser Thir Wall Luell 485 490 495

Asn Luell Asp Phe Pro Ala Gly Wall Wall Pro Wall Thir Thir Wall SOO 505

Thir Ala Glu Asp Asp Ala Glin Met Glu Luell Tyr Gly Phe Gly 515 525

Asp Ile Trp Asp Ile Ile Lell Ala Met Lys Asn Ser Wall Gly 53 O 535 54 O

Lell Pro Wall Ala Wall Glin Wall Ala Luell Pro Trp Glin Glu Glu Luell 5.45 550 555 560

Luell Arg Phe Met Arg Glu Wall Glu Glin Luell Met Thir Pro Glin Lys 565 st O sts

Glin Pro Ser

SEQ ID NO 32 LENGTH: 236 TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 32 Met Ser Ser Ser Ala Ala Ser Pro Leu Phe Ala Pro Gly Glu Asp Cys 1. 5 15 Gly Pro Ala Trp Arg Ala Ala Pro Ala Ala Tyr Asp Thir Ser Asp Thr 25 US 7,972,785 B2 141 142 - Continued

His Luell Glin Ile Lieu. Gly Pro Wall Met Glu Arg Trp Glu. Thr Pro 35 4 O 45

Met His Ser Luell Ala Ala Ala Ala Ala Ser Arg Gly Gly Arg Wall SO 55 6 O

Lell Glu Wall Gly Phe Gly Met Ala Ile Ala Ala Ser Arg Wall Glin Glin 65 70

Ala Pro Ile Lys Glu His Trp Ile Ile Glu Cys Asn Asp Gly Wall Phe 85 90 95

Glin Arg Luell Glin Asn Trp Ala Luell Lys Glin Pro His Wall Wall Pro 1OO 105 11 O

Lell Gly Lieu. Trp Glu Glu Glu Ala Pro Thir Lell Pro Asp Gly His 115 12 O 125

Phe Asp Gly Ile Lieu. Asp Thir Pro Luell Ser Glu Glu Thir Trp 13 O 135 14 O

His Thir His Glin Phe Asn Phe Ile Thir His Ala Phe Arg Luell Luell 145 150 155 160

Pro Gly Gly Ile Lell Thir Asn Luell Thir Ser Trp Gly Glu 1.65 17O 17s

Lell Met Ser Lys Thir Asp Ile Thir Ala Met Phe Glu Glu Thir 18O 185 19 O

Glin Wall Pro Ala Lieu. Lell Glu Ala Gly Phe Glin Arg Glu Asn Ile 195 2OO

Thir Glu Wall Met Ala Lell Wall Pro Pro Ala Asp Cys Arg Ala 21 O 215 22O

Phe Pro Glin Met Ile Thir Pro Luell Wall Thir Lys His 225 23 O 235

<210s, SEQ ID NO 33 &211s LENGTH: 440 212. TYPE : PRT &213s ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 33

Met Val His Gly Tyr Gly Wall Glin Phe Glin Asn Trp Ala Lys Thir 1. 5 15

Tyr Gly Cys Ser Pro Glu Wall Tyr Glin Pro Thir Ser Wall Glu Glu 2O 25

Wall Arg Glu Wall Lieu. Ala Lell Ala Arg Glu Glin Lys Wall 35 4 O 45

Wall Wall Gly Gly Gly His Ser Pro Ser Asp Ile Ala Thir Asp Gly SO 55 6 O

Phe Met Ile His Met Gly Met Asn Arg Wall Lell Glin Wall Asp Lys 65 70

Glu Glin Ile Thir Wall Glu Ala Gly Ile Lell Lell Ala Asp Luell 85 90 95

His Pro Glin Lieu. Asp Glu His Gly Luell Ala Met Ser Asn Luell Gly Ala 1OO 105 11 O

Wall Ser Asp Wall. Thir Wall Ala Gly Wall Ile Gly Ser Gly Thir His Asn 115 12 O 125

Thir Gly Ile Llys His Gly Ile Luell Ala Thir Glin Wall Wall Ala Luell Thir 13 O 135 14 O

Lell Met Thir Ala Asp Gly Glu Wall Luell Glu Cys Ser Glu Ser Arg Asn 145 150 155 160

Ala Asp Wall Phe Glin Ala Ala Arg Wall His Luell Gly Luell Gly Ile 1.65 17O 17s US 7,972,785 B2 143 144 - Continued

Ile Luell Thir Wall Thir Lell Glin Wall Pro Glin Phe His Luell Glin Glu 18O 185 19 O

Thir Ser Phe Pro Ser Thir Lell Lys Glu Wall Luell Asp Asn Luell Asp Ser 195 2O5

His Luell Arg Ser Glu Tyr Phe Arg Phe Luell Trp Phe Pro His Thir 21 O 215

Glu Asn Wall Ser Ile Ile Glin Asp His Thir Asn Ala Pro Ser 225 23 O 235 24 O

Ser Ala Ser Asn Trp Phe Trp Asp Ala Ile Gly Phe Luell Luell 245 250 255

Glu Phe Luell Luell Trp Thir Ser Thir Tyr Luell Pro Lell Wall Gly Trp 26 O 265 27 O

Ile Asn Arg Phe Phe Phe Trp Met Luell Phe ASn Lys Glu Ser 285

Ser Asn Luell Ser His Ile Phe Thir Glu Cys Arg Phe Glin 29 O 295 3 OO

His Wall Glin Asp Trp Ala Ile Pro Arg Glu Lys Thir Glu Ala Luell 3. OS 310 315

Lell Glu Luell Ala Met Lell Glu Ala His Pro Wall Wall Ala His 3.25 330 335

Pro Wall Glu Wall Arg Phe Thir Arg Gly Asp Asp Ile Luell Luell Ser 34 O 345 35. O

Pro Phe Glin Arg Asp Ser Cys Met ASn Ile Ile Met 355 360 365

Pro Tyr Gly Asp Wall Pro Arg Luell Asp Trp Lell Ala Glu 37 O 375

Thir Ile Met Lys Phe Gly Gly Arg Pro His Trp Ala Ala His 385 390 395 4 OO

Asn Thir Arg Lys Asp Phe Glu Glu Met Pro Thir Phe His Lys 4 OS 415

Phe Asp Ile Arg Glu Lys Luell Asp Pro Thir Gly Met Phe Luell Asn 42O 425 43 O

Ser Luell Glu Wall Phe Tyr 435 44 O

<210s, SEQ ID NO 34 &211s LENGTH: 335 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 34

Met Ala Ala Ser Lell Gly Phe Arg Gly Ala Ala Ser Gly Luell Arg Tyr 1. 5 1O 15

Trp Ser Gly Arg Arg Arg Pro Wall Gly Ser Luell Ala Ala Wall Ser 25 3O

Arg Ser Met Ala Ser Thir Pro Wall Gly Phe Ile Gly Luell Gly Asn 35 4 O 45

Met Gly Asn Pro Met Ala Lys Asn Luell Ile His Gly Tyr Pro Luell SO 55 6 O

Ile Luell Tyr Asp Wall Phe Pro Asp Wall Lys Glu Phe Glu Ala 65 70 7s

Gly Glu Glin Wall Ala Ser Ser Pro Ala Asp Wall Ala Glu Ala Asp 85 90 95

Arg Ile Ile Thir Met Lell Pro Ser Ser Met ASn Ser Ile Glu Wall Tyr 1OO 105 11 O US 7,972,785 B2 145 146 - Continued

Ser Gly Ala Asn Gly Ile Lell Lys Wall Lys Lys Gly Ser Luell Luell 115 12 O 125

Ile Asp Ser Ser Thir Ile Asp Pro Ser Wall Ser Lys Glu Luell Ala 13 O 135 14 O

Glu Wall Glu Met Gly Ala Wall Phe Met Asp Ala Pro Wall Ser Gly 145 150 155 160

Gly Wall Gly Ala Ala Arg Ser Gly Asn Luell Thir Phe Met Wall Gly Gly 1.65 17O 17s

Wall Glu Asn Glu Phe Ala Ala Ala Glin Glu Luell Lell Gly Cys Met Gly 18O 185 19 O

Ser Asn Wall Luell Tyr Gly Ala Wall Gly Ser Gly Glin Ser Ala 195

Ile Cys Asn Asn Met Lell Lell Ala Ile Ser Met Ile Gly Thir Ala Glu 21 O 215

Ala Met Asn Luell Gly Ile Arg Ser Gly Luell Asp Pro Luell Luell Ala 225 23 O 235 24 O

Ile Luell Asn Met Ser Ser Gly Arg Cys Trp Ser Ser Asp Thir 245 250 255

Asn Pro Wall Pro Gly Wall Met Asp Gly Wall Pro Ser Ser Asn Asn 26 O 265 27 O

Glin Gly Gly Phe Gly Thir Thir Luell Met Ala Asp Lell Gly Luell Ala 285

Glin Asp Ser Ala Thir Ser Thir Thir Pro Ile Lell Lell Gly Ser Wall 29 O 295 3 OO

Ala His Glin Ile Tyr Arg Met Met Ser Lys Gly Ser Lys 3. OS 310 315 32O

Asp Phe Ser Ser Wall Phe Glin Luell Arg Glu Glu Glu Thir Phe 3.25 330 335

<210s, SEQ ID NO 35 &211s LENGTH: 298 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 35

Met Glu Glu Lys Glin Ile Lell Wall Gly Luell Wall Wall Luell Asp Ile 1. 5 15

Ile Asn Wall Wall Asp Pro Glu Glu Asp Thir Asp Arg Arg 25

Lell Ser Glin Arg Trp Glin Arg Gly Gly Asn Ala Ser Asn Ser Thir 35 4 O 45

Wall Luell Ser Luell Lell Gly Ala Arg Ala Phe Met Gly Ser Luell Ala SO 55 6 O

His Gly His Wall Ala Asp Phe Luell Wall Ala Asp Phe Arg Arg Arg Gly 65 70

Wall Asp Wall Ser Glin Wall Ala Trp Glin Ser Glin Gly Asp Thir Pro Cys 85 90 95

Ser Ile Wall Asn Asn Ser Asn Gly Ser Arg Thir Ile Ile Luell 105 11 O

Asp Thir Asn Lell Pro Asp Wall Ser Ala Lys Asp Phe Glu Wall 115 12 O 125

Asp Luell Thir Arg Phe Trp Ile His Ile Glu Gly Arg Asn Ala Ser 13 O 135 14 O

Glu Glin Wall Lys Met Lell Glin Arg Ile Glu Glin Asn Ala Thir Glin 145 150 155 160 US 7,972,785 B2 147 148 - Continued

Pro Luell Glin Glin Lys Wall Arg Wall Ser Wall Glu Ile Glu Lys Pro Arg 1.65 17O 17s

Glu Glu Luell Phe Glin Lell Phe Gly Tyr Gly Glu Wall Wall Phe Wall Ser 18O 185 19 O

Asp Wall Ala Lys His Lell Gly Phe Arg Ser Ala Gly Glu Ala Lieu 195

Gly Luell Ser Arg Wall Gly Ala Thir Lell Ile Cys Ala 21 O 215 22O

Trp Ala Glu Glu Gly Ala Asp Ala Luell Gly Pro Asp Gly Glin Lieu. Luell 225 23 O 235 24 O

His Ser Asp Ala Phe Pro Pro Pro Arg Wall Wall Asp Thir Luell Gly Ala 245 250 255

Gly Asp Thir Phe Asn Ala Ser Wall Ile Phe Ser Lell Ser Lys Gly Asn 26 O 265 27 O

Ser Met Glin Glu Ala Lell Arg Phe Gly Glin Wall Ala Gly 27s 28O 285

Gly Luell Glin Gly Phe Asp Gly Ile Wall 29 O 295

SEQ ID NO 36 LENGTH: 464 TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 36

Met Glu Pro Ser Pro Lell Glu Luell Pro Wall Asp Ala Wall Arg Arg Ile 1. 5 15

Ala Ala Glu Luell Asn Asp Pro Thir Asp Glu Arg Wall Ala Lieu. Arg 2O 25

Lell Asp Glu Glu Asp Lell Lys Arg Phe Asp Cys Phe Tyr Ile 35 4 O 45

Pro Lys Met Arg Asp Lell Pro Ser Ile Asp Luell Ser Lell Wall Asn. Glu SO 55 6 O

Asp Asp Asn Ala Ile Tyr Phe Luell Gly Asn Ser Lell Gly Luell Gln Pro 65 70 8O

Met Wall Thir Lell Glu Glu Glu Luell Asp Trp Ala Lys 85 90 95

Ile Gly Ala Tyr Gly His Glu Wall Gly Arg Pro Trp Ile Ile Gly 105 11 O

Asp Glu Ser Ile Wall Thir Lell Met Asp Ile Wall Gly Ala His Glu 115 12 O 125

Glu Ile Ala Lell Met Asn Ala Luell Thir Wall Asn Lell His Lieu. Luell 13 O 135 14 O

Lell Luell Ser Phe Phe Lys Pro Thir Pro Arg His Ile Lieu. Luell 145 150 155 160

Glu Ala Ala Phe Pro Ser Asp His Tyr Ala Ile Glu Ser Glin Ile 1.65 17O 17s

Glin Luell His Gly Lell Asp Wall Glu Lys Ser Met Arg Met Ile Llys Pro 18O 185 19 O

Arg Glu Gly Glu Glu Thir Lell Arg Met Glu Asp Ile Lell Glu Wall Ile 195

Glu Lys Glu Gly Asp Ser Ile Ala Wall Wall Luell Phe Ser Gly Lieu. His 21 O 215 22O

Phe Thir Gly Glin Lell Phe Asn Ile Pro Ala Ile Thir Glin Ala Gly 225 23 O 235 24 O US 7,972,785 B2 149 150 - Continued

His Ala Gly Cys Phe Wall Gly Phe Asp Luell Ala His Ala Wall Gly 245 250 255

Asn Wall Glu Luell His Lell His Asp Trp Asp Wall Asp Phe Ala Cys Trp 26 O 265 27 O

Ser Tyr Tyr Lell Asn Ser Gly Ala Gly Gly Lell Ala Gly Ala 27s 28O 285

Phe Ile His Glu Lys His Ala His Thir Ile Pro Ala Luell Wall Gly 29 O 295 3 OO

Trp Phe Gly His Glu Lell Ser Thir Arg Phe ASn Met Asp Asn Luell 3. OS 310 315

Glin Luell Ile Pro Gly Wall Asn Gly Phe Arg Ile Ser Asn Pro Pro Ile 3.25 330 335

Lell Luell Wall Cys Ser Lell His Ala Ser Luell Glu Ile Phe Glin Glin Ala 34 O 345 35. O

Thir Met Thir Ala Lell Arg Arg Lys Ser Ile Luell Lell Thir Gly Luell 355 360 365

Glu Tyr Luell Luell Lys His Tyr His Gly Gly ASn Asp Thir Glu Asn 37 O 375

Arg Pro Wall Wall Asn Ile Ile Thir Pro Ser Arg Ala Glu Glu Arg Gly 385 390 395 4 OO

Glin Luell Thir Lell Thir Phe Ser Ile Ser Gly Wall Phe 4 OS 415

Glu Luell Glu Lys Arg Gly Wall Wall Cys Asp Arg Glu Pro Glu 425 43 O

Ile Arg Wall Ala Pro Wall Pro Luell Asn Ser Phe His Asp Wall 435 44 O 445

Phe Ile Arg Lell Lell Thir Ala Ile Luell Asp Ser Thir Glu Arg Asn 450 45.5 460

<210s, SEQ ID NO 37 &211s LENGTH: 11.78 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 37

Met Leu Lys Phe Glin Thir Wall Arg Gly Gly Luell Arg Lell Luell Gly Wall 1. 5 15

Arg Arg Ser Ser Thir Ala Pro Wall Ala Ser Pro Asn Wall Arg Arg Luell 2O 25

Glu Lys Pro Ile Wall Met Wall Ala Asn Arg Gly Glu Ile 35 4 O 45

Ala Ile Arg Wall Phe Arg Ala Thir Glu Luell Gly Ile Arg Thir Wall SO 55 6 O

Ala Wall Ser Glu Glin Asp Thir Gly Glin Met His Arg Glin Ala 65 70

Asp Glu Ala Lell Ile Gly Arg Gly Luell Ala Pro Wall Glin Ala Tyr 85 90 95

Lell His Ile Pro Asp Ile Ile Wall Ala Glu Asn Gly Wall Asp 1OO 105 11 O

Ala Wall His Pro Gly Gly Phe Luell Ser Glu Arg Ala Asp Phe Ala 115 12 O 125

Glin Ala Glin Asp Ala Gly Wall Arg Phe Ile Gly Pro Ser Pro Glu 13 O 135 14 O

Wall Wall Arg Met Gly Asp Wall Glu Ala Arg Ala Ile Ala Ile 145 150 155 160 US 7,972,785 B2 151 152 - Continued

Ala Ala Gly Wall Pro Wall Wall Pro Gly Thir ASn Ser Pro Ile Asn Ser 1.65 17s

Lell His Glu Ala His Glu Phe Ser Asn Thir Gly Phe Pro Ile Ile 18O 185 19 O

Phe Ala Ala Tyr Gly Gly Gly Gly Arg Gly Met Arg Wall Wall His 195 2O5

Ser Tyr Glu Glu Lell Glu Glu Asn Tyr Thir Arg Ala Ser Glu Ala 21 O 215

Lell Ala Ala Phe Gly Asn Gly Ala Luell Phe Wall Glu Phe Ile Glu 225 23 O 235 24 O

Pro Arg His Ile Glu Wall Glin Ile Luell Gly Asp Glin Tyr Gly Asn 245 250 255

Ile Luell His Luell Tyr Glu Arg Asp Cys Ser Ile Glin Arg Arg His Glin 26 O 265 27 O

Wall Wall Glu Ile Ala Pro Ala Thir His Luell Asp Pro Glin Luell Arg 285

Ser Arg Luell Thir Ser Asp Ser Wall Luell Ala Lys Glin Wall Gly Tyr 29 O 295 3 OO

Glu Asn Ala Gly Thir Wall Glu Phe Luell Wall Asp His Gly His 3. OS 310 315

Phe Ile Glu Wall Asn Ser Arg Luell Glin Wall Glu His Thir Wall Thir 3.25 330 335

Glu Glu Ile Thir Asp Wall Asp Luell Wall His Ala Glin Ile His Wall Ser 34 O 345 35. O

Glu Gly Arg Ser Lell Pro Asp Luell Gly Luell Arg Glin Glu Asn Ile Arg 355 360 365

Ile Asn Gly Ala Ile Glin Arg Wall Thir Thir Glu Asp Pro Ala 37 O 375

Arg Ser Phe Glin Pro Asp Thir Gly Arg Ile Glu Wall Phe Arg Ser Gly 385 390 395 4 OO

Glu Gly Met Gly Ile Arg Lell Asp Asn Ala Ser Ala Phe Glin Gly Ala 4 OS 415

Wall Ile Ser Pro His Asp Ser Luell Luell Wall Wall Ile Ala His 425 43 O

Gly Asp His Pro Thir Ala Ala Thir Met Ser Arg Ala Luell Ala 435 44 O 445

Glu Phe Arg Wall Arg Gly Wall Thir Asn Ile Pro Phe Luell Glin Asn 450 45.5 460

Wall Luell Asn Asn Glin Glin Phe Luell Ala Gly Ile Wall Asp Thir Glin Phe 465 470

Ile Asp Glu Asn Pro Glu Lell Phe Glin Luell Arg Pro Ala Glin Asn Arg 485 490 495

Ala Glin Luell Lell His Luell Gly His Wall Met Wall Asn Gly Pro SOO 505

Thir Thir Pro Ile Pro Wall Wall Ser Pro Ser Pro Wall Asp Pro Ile 515 525

Wall Pro Wall Wall Pro Ile Gly Pro Pro Pro Ala Gly Phe Arg Asp Ile 53 O 535 54 O

Lell Luell Arg Glu Gly Pro Glu Gly Phe Ala Arg Ala Wall Arg Asn His 5.45 550 555 560

Glin Gly Luell Luell Lell Met Asp Thir Thir Phe Arg Asp Ala His Glin Ser 565 st O sts

Lell Luell Ala Thir Wall Arg Thir His Asp Luell Ile Ala Pro 58O 585 59 O US 7,972,785 B2 153 154 - Continued

Wall Ala His Asn Phe Asn Asn Luell Phe Ser Ile Glu Asn Trp Gly 595 605

Gly Ala Thir Phe Asp Wall Ala Met Arg Phe Luell Tyr Glu Cys Pro Trp 610 615

Arg Arg Luell Glin Glu Lell Arg Glu Luell Ile Pro Asn Ile Pro Phe Glin 625 630 635 64 O

Met Luell Luell Arg Gly Ala Asn Ala Wall Gly Tyr Thir Asn Pro Asp 645 650 655

Asn Wall Wall Phe Phe Glu Wall Ala Glu Asn Gly Met Asp 660 665 67 O

Wall Phe Arg Ile Phe Asp Ser Luell Asn Tyr Luell Pro Asn Met Luell Luell 675 685

Gly Met Glu Ala Ala Gly Ser Ala Gly Gly Wall Wall Glu Ala Ala Ile 69 O. 695 7 OO

Ser Thir Gly Asp Wall Ala Asp Pro Ser Arg Thir Ser Luell 7 Os

Glu Tyr Met Gly Lell Ala Glu Glu Luell Wall Arg Ala Gly Thir His 72 73 O 73

Ile Luell Ile Lys Asp Met Ala Gly Luell Luell Pro Ala Ala 740 74. 7 O

Thir Met Luell Wall Ser Ser Lell Arg Asp Arg Phe Pro Asp Luell Pro Luell 760 765

His Ile His Thir His Asp Thir Ser Gly Ser Gly Wall Ala Ala Met Luell 770 775

Ala Ala Glin Ala Gly Ala Asp Wall Wall Asp Wall Ala Wall Asp Ser 79 O 79.

Met Ser Gly Met Thir Ser Glin Pro Ser Met Gly Ala Lell Wall Ala Cys 805 810 815

Thir Gly Thir Pro Lell Asp Thir Glu Wall Pro Lell Glu Arg Wall Phe 825 83 O

Asp Ser Glu Tyr Trp Glu Gly Ala Arg Gly Lell Tyr Ala Ala Phe 835 84 O 845

Asp Cys Thir Ala Thir Met Lys Ser Gly Asn Ser Asp Wall Glu Asn 850 855 860

Glu Ile Pro Gly Gly Glin Tyr Thir Asn Luell His Phe Glin Ala His Ser 865

Met Gly Luell Gly Ser Phe Glu Wall Ala Wall Glu 885 890 895

Ala Asn Glin Met Lell Gly Asp Luell Ile Wall Thir Pro Ser Ser 9 OO 905 91 O

Ile Wall Gly Asp Lell Ala Glin Phe Met Wall Glin Asn Gly Luell Ser Arg 915 92 O 925

Ala Glu Ala Glu Ala Glin Ala Glu Glu Luell Ser Phe Pro Arg Ser Wall 93 O 935 94 O

Wall Glu Phe Luell Glin Gly Tyr Ile Gly Ile Pro His Gly Gly Phe Pro 945 950 955 96.O

Glu Pro Phe Arg Ser Wall Luell Lys Asp Luell Pro Arg Ile Glu Gly 965 97.

Arg Pro Gly Ala Ser Lell Pro Pro Luell Asn Luell Lys Glu Luell Glu Lys 985 99 O

Asp Luell Ile Asp Arg His Gly Glu Glu Val Thr Pro Glu Asp Val Lieu. 995 1OOO

Ser Ala Ala Met Tyr Pro Asp Val Phe Ala Glin Phe Lys Asp Phe US 7,972,785 B2 156 - Continued

O15 O2O

Thir Thir Phe Gly Pro Lieu. Asp Ser Lieu. Asn. Thir Arg Lieu. Phe O3 O O35

Lell Gly Pro Ile Ala Glu Glu Phe Glu Wall Glu Lieu. Glu O45 OSO

Arg Thir Luell His Ile Lys Ala Lieu Ala Val Ser Asp Lieu. O6 O O65

Asn Ala Gly Glin Arg Glin Val Phe Phe Glu Lieu. Asn Gly Glin O7 O8O

Lell Ser Ile Luell Val Lys Asp Thr Glin Ala Met Lys Glu Met O9 O O95

His His Pro Ala Lieu Lys Asp Wall Lys Gly Glin e Gly 105 11 O

Ala Met Pro Llys Val Ile Asp Val Llys Val Ala Ala Gly 12 O 125

Ala Wall Wall Gly Glin Pro Leu. Cys Val Lieu Ser Ala Met 135 14 O

Glu Thir Wall Val Thr Ser Pro Met Glu Gly Thr e Arg 15 O 155

His Wall Thir Lys Asp Met Thr Lieu. Glu Gly Asp Asp Lieu. 1.65 17 O

Ile Glu Ile Glu

<210s, SEQ ID NO 38 &211s LENGTH: 4 O 7 212. TYPE : PRT <213> ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 38

Met Arg Trp Phe Ala Lieu. Lieu Lys Asn Ala Ser Lieu Ala Gly Ala 1. 1O 15

Pro Lys Tyr Ile His Phe Ser Llys Phe Ser Pro Ser Pro Leu Ser 25 3O

Met Glin Phe Lell Asp Phe Gly Ser Ser Asn Ala Cys Glu Lys Thr 35 4 O 45

Ser Thir Phe Lell Arg Glin Glu Lieu Pro Val Arg Lieu Ala Asn. Ile 55 6 O

Met Glu Ile Asn Lieu Lleu Pro Asp Arg Val Lieu. Ser Thr Pro Ser 65 70 7s 8O

Wall Glin Luell Wall Glin Ser Trp Tyr Val Glin Ser Lieu Lleu. Asp Ile Met 85 90 95

Glu Phe Luell Asp Lys Asp Pro Glu Asp His Arg Thr Lieu. Ser Glin Phe 105 11 O

Thir Asp Ala Luell Wall Thir Ile Arg Asn Arg His Asn Asp Val Val Pro 115 12 O 125

Thir Met Ala Glin Gly Val Lieu. Glu Tyr Lys Asp Thr Tyr Gly Asp Asp 13 O 135 14 O

Pro Wall Ser Asn Glin Asn. Ile Glin Tyr Phe Lieu. Asp Arg Phe Tyr Lieu. 145 150 155 160

Ser Arg Ile Ser Ile Arg Met Lieu. Ile Asin Gln His Thr Lieu. Ile Phe 1.65 17O 17s

Asp Gly Ser Thir Asn Pro Ala His Pro Llys His Ile Gly Ser Ile Asp 18O 185 19 O

Pro Asn Ser Wall Ser Asp Val Val Lys Asp Ala Tyr Asp Met Ala US 7,972,785 B2 157 158 - Continued

195 2OO 2O5

Luell Luell Tyr Met Ala Ser Pro Asp Luell Glu Ile 21 O 215 22O

Glin Glu Wall Asn Ala Thir Asn Ala Thir Glin Pro Ile His Met Wall Tyr 225 23 O 235 24 O

Wall Pro Ser His Lieu. His Met Luell Phe Glu Lell Phe Asn Ala 245 250 255

Met Arg Ala Thir Wall Glu Ser His Glu Ser Ser Lell Thir Luell Pro Pro 26 O 265 27 O

Ile Ile Met Wall Ala Lell Gly Glu Glu Asp Lell Ser Ile Met 285

Ser Asp Arg Gly Gly Gly Wall Pro Luell Arg Ile Glu Arg Luell Phe 29 O 295 3 OO

Ser Met Tyr Ser Thir Ala Pro Thir Pro Glin Pro Gly Thir Gly Gly 3. OS 310 315

Thir Pro Luell Ala Gly Phe Gly Gly Luell Pro Ile Ser Arg Luell Tyr 3.25 330 335

Ala Phe Glin Gly Asp Luell Glin Luell Phe Ser Met Glu Gly Phe 34 O 345 35. O

Gly Thir Asp Ala Wall Ile Luell Ala Luell Ser Thir Asp Ser Wall 355 360 365

Glu Arg Luell Pro Wall Asn Ser Ala Trp Arg His Glin Thir 37 O 375

Ile Glin Glu Ala Gly Asp Trp Wall Pro Ser Thir Glu Pro Asn 385 390 395 4 OO

Thir Ser Thir Wall Ser 4 OS

<210s, SEQ ID NO 39 &211s LENGTH: 362 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 39

Met Glu Wal His ASn Wall Ser Ala Pro Phe ASn Phe Ser Luell Pro Pro 1. 5 15

Gly Phe Gly His Arg Ala Thir Asp Lys Ala Luell Ser Ile Ile Luell Wall 2O 25

Lell Met Luell Lieu. Luell Ile Met Luell Ser Luell Gly Thir Met Glu Phe 35 4 O 45

Ser Lys Ile Lys Ala His Lell Trp Pro Gly Wall Ile Wall Ala SO 55 6 O

Lell Wall Ala Glin Phe Gly Ile Met Pro Luell Ala Ala Phe Luell Luell Gly 65 70 7s

Ile Phe His Lieu. Ser Asn Ile Glu Ala Luell Ala Ile Luell Ile Cys 85 90 95

Ser Pro Gly Gly Asn Luell Ser Asn Luell Phe Thir Luell Ala Met 1OO 105 11 O

Gly Asp Met Asn Lell Ser Ile Wall Met Thir Thir Cys Ser Ser Phe 115 12 O 125

Ser Ala Luell Gly Met Met Pro Luell Luell Luell Wall Ser Gly 13 O 135 14 O

Ile Asp Gly Asp Lell Asp Wall Pro Gly Ile Met 145 150 155 160

Ile Ser Luell Wall Ile Wall Lell Ile Pro Cys Thir Ile Gly Ile Wall Luell US 7,972,785 B2 159 160 - Continued

1.65 17O 17s

Ser Arg Pro His Wall Pro Tyr Ile Lell Gly Gly Met 18O 185 19 O

Ile Ile Thir Phe Lell Lell Ser Wall Ala Wall Thir Ala Lell Ser Wall Ile 195

Asn Wall Gly Asn Ser Ile Met Phe Wall Met Thir Pro His Luell Luell Ala 21 O 215 22O

Thir Ser Ser Luell Met Pro Phe Ser Gly Phe Luell Met Gly Ile Luell 225 23 O 235 24 O

Ser Ala Luell Phe Glin Lell Asn Pro Ser Cys Arg Arg Thir Ile Ser Met 245 250 255

Glu Thir Gly Phe Glin Asn Ile Glin Luell Ser Thir Ile Luell Asn Wall 26 O 265 27 O

Thir Phe Pro Pro Glu Wall Ile Gly Pro Luell Phe Phe Phe Pro Luell Luell 27s 285

Met Ile Phe Glin Lell Ala Glu Gly Luell Luell Ile Ile Ile Ile Phe 29 O 295 3 OO

Arg Glu Lys Ile Pro Pro Asp Glin Thir Ile Thir 3. OS 310 315

Ala Ala Ala Thir Glu Asp Ala Thir Pro Ala Ala Luell Glu Lys 3.25 330 335

Gly Thir His Asn Gly Asn Ile Pro Pro Luell Glin Pro Gly Pro Ser Pro 34 O 345 35. O

Asn Gly Luell Asn Ser Gly Glin Met Ala Asn 355 360

<210s, SEQ ID NO 4 O &211s LENGTH: 429 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 4 O

Met Ala Ala Glin Gly Gly Arg Thir Wall Ile Phe Thir Ala 1. 5 15

Met Phe Gly Gly Tyr Ser Lell Tyr Phe ASn Arg Thir Phe Ser 25

Phe Wall Met Pro Ser Lell Wall Asp Glu Ile Ala Lell Asp Asp Asp 35 4 O 45

Lell Gly Luell Ile Thir Ser Ser Glin Ser Ala Ala Tyr Ala Ile Ser Lys SO 55 6 O

Phe Wall Ser Gly Wall Lell Ser Asp Glin Met Ser Ala Arg Trp Luell Phe 65 70

Ser Ser Gly Luell Lell Lell Wall Gly Luell Wall ASn Wall Wall Phe Ser Trp 85 90 95

Ser Ser Thir Wall Thir Ala Phe Ala Ala Luell Trp Phe Lell Asn Gly Luell 105 11 O

Ala Glin Gly Luell Gly Trp Pro Pro Gly Ile Lell Arg Trp 115 12 O 125

Phe Glu Pro Ser Glin Phe Gly Thir Trp Trp Ala Wall Lell Ser Thir Ser 13 O 135 14 O

Met Asn Luell Ala Gly Ser Lell Gly Pro Ile Luell Ala Thir Ile Luell Ala 145 150 155 160

Glin Ser Ser Trp Arg Ser Thir Luell Ala Luell Ser Gly Ser Luell Cys 1.65 17O 17s

Wall Wall Wall Ser Phe Phe Luell Luell Luell Ile His Asn Glu Pro Ala US 7,972,785 B2 161 162 - Continued

18O 185 19 O

Asp Wall Gly Luell Arg Asn Lell Asp Pro Ala Pro Ser Lys Gly Lys 195

Gly Ser Ser Glu Glu Ser Thir Luell Glin Asp Lell Lell Luell Ser Pro 21 O 215

Tyr Luell Trp Wall Lell Ser Thir Gly Luell Wall Wall Phe Gly Wall Lys 225 23 O 235 24 O

Thir Thir Asp Trp Gly Glin Phe Phe Luell Ile Glin Glu Arg Gly 245 250 255

Glin Ser Ala Luell Wall Gly Ser Ser Tyr Ile Ser Ala Lell Glu Wall Gly 26 O 265 27 O

Gly Luell Wall Gly Ser Ile Ala Ala Gly Luell Ser Asp Arg Ala Met 285

Ala Lys Ala Gly Lell Ser Wall Gly Asn Pro Arg His Ser Luell Luell 29 O 295 3 OO

Lell Luell Met Met Ala Gly Met Ala Ala Ser Met Phe Lell Phe Arg Wall 3. OS 310 315

Thir Wall Thir Ser Asp Ser Pro Ile Trp Ile Lell Wall Luell Gly Ala 3.25 330 335

Wall Phe Gly Phe Ser Ser Gly Pro Ile Ala Lell Phe Gly Wall Ile 34 O 345 35. O

Ala Asn Glu Ser Ala Pro Pro Asn Luell Gly Thir Ser His Ala Ile 355 360 365

Wall Gly Luell Met Ala Asn Wall Gly Gly Phe Luell Ala Gly Luell Pro Phe 37 O 375

Ser Thir Ile Ala Lys His Ser Trp Ser Thir Ala Phe Trp Wall Ala 385 390 395 4 OO

Glu Wall Ile Ile Ala Ser Thir Wall Wall Phe Phe Lell Luell Arg Asn 4 OS 41O 415

Ile Arg Thir Lys Met Gly Arg Wall Ser Ala Glu 42O 425

<210s, SEQ ID NO 41 &211s LENGTH: 463 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 41

Met Lieu. Cys Ser Glu Lell Glu Thir Gly Ala Trp Gly Ala Luell Ala 1. 5

Glu Luell Gly Ala Ser Glu Ala Ala Ser Wall Ser Wall Asp Glin Ala 25

Gly Ser Ala Luell Lell Gly Lell Glu Ser Glu Ala Ala Trp Ala Ser 35 4 O 45

Lell Phe Ala Ser Arg Pro Ala Ala Gly Ala Ser Wall Phe Luell Ser SO 55 6 O

Pro Pro Ser Trp Gly Ala Arg Ser Gly Ala Arg Wall Pro Ser Luell 65 70

Gly Ala Arg Luell Ala Gly Lell Gly Gly Luell Wall Ala His Arg Pro 85 90

Gly Pro Gly Arg Lell Lell Lell Arg His Trp Pro Gly Lell Gly Luell 105 11 O

Lell Arg Gly Ala Lell Ala Lell Luell Gly Luell Gly Lell Gly Arg Luell Luell 115 12 O 125

Lell Thir Gly Arg Lell Lell Wall Gly Luell Luell Phe Lell Ser Pro Luell US 7,972,785 B2 163 164 - Continued

13 O 135 14 O

Pro Ile Trp Ser Wall Lell Gly Luell Luell Ala Lell Lell Asp Luell Ser 145 150 155 160

Gly Wall Gly Gly Arg Lell Gly Phe Ser Luell Luell Trp Gly Pro Luell Phe 1.65 17O 17s

Lell Gly Ala Arg Ala Phe Ser Luell Luell Wall Phe Wall Lell Gly Luell Luell 18O 185 19 O

Lell Ile His Ile Wall Thir Lell Luell Ala Glin Luell Ala Ser His His Gly 195

Wall Pro Luell Lieu. Arg Gly Ala Asp Glu Ser Ser Lell His Phe Phe 21 O 215 22O

Lys Luell Tyr Lieu. Luell Pro Lell Pro His Asn Lys Lell Trp Ala Thir Asn 225 23 O 235 24 O

Glin Thir Pro Ile Asn Glu Arg Thir Glu Met Wall Ser Phe Arg Glin 245 250 255

Lell Glin Ala Ala Arg Ile Glin Gly Lys Arg Trp Thir His Pro Glu 26 O 265 27 O

Gly Glin Luell Gly Ser Ser Asn Ile Wall Pro Lell Thir His Luell Ser 285

Ser Ser Pro Lell Tyr Wall Ala Arg ASn Ala Asp Met 29 O 295 3 OO

Wall Ser Tyr His Phe Arg Met Ser Glin Wall Lell Pro Asn Pro 3. OS 310 315

Gly Thir Trp Asn. Glu Tyr Phe Glu Thir Phe Ile Asn Gly Wall Ser 3.25 330 335

Gly Ser Trp Phe Asp His Wall Lys Gly Trp Trp Glu Ile Arg Asp 34 O 345 35. O

Glin Ile Lieu. Phe Lell Phe Glu Asp Met Lys Arg Asp Pro 355 360 365

Arg Glu Ile Glin Wall Met Glin Phe Met Gly Lys Asn Luell Asp 37 O 375

Glu Glu Wall Val Asp Lys Ile Wall Luell Glu Thir Ser Phe Glu Met 385 390 395 4 OO

Asp Asn Pro Leu. Thir Asn Phe Ser Thir Ile Pro Thir Ile Met 4 OS 415

Glin Ser Ile Ser Pro Phe Met Arg Lys Gly Ile Wall Gly Asp Trp 42O 425 43 O

Asn His Phe Thir Wall Ala Glin Asn Glu Arg Phe Asp Glu Ile Tyr 435 44 O 445

Glu Glin Met Asp Gly Thir Ser Luell Asn Phe Cys Met Glu Luell 450 45.5 460

SEQ ID NO 42 LENGTH: 161 TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 42

Met Asp Glin Glu Ala Wall Gly Asn Wall Wall Luell Lell Ala Ile Wall Thir 1. 5 1O 15

Lell Ile Ser Wall Wall Glin Asn Ala Phe Phe Ala His Wall Glu Luell 2O 25 3O

Glu Ser Lys Ala Glin Ser Gly Arg Ser Phe Glin Arg Thir Gly Thir Luell 35 4 O 45

Ala Phe Glu Arg Val Tyr Thir Ala Asn Glin ASn Wall Asp Ala Tyr US 7,972,785 B2 165 166 - Continued

SO 55 6 O

Pro Thir Phe Luell Wall Wall Lieu. Trp Thir Ala Gly Lell Lell Ser Glin 65 70

Wall Pro Ala Ala Phe Ala Gly Lieu. Met Tyr Luell Phe Wall Arg Glin 85 90 95

Phe Wall Gly Tyr Lell Gly Glu Arg Thir Glin Ser Thir Pro Gly Tyr 105 11 O

Ile Phe Gly Lys Ile Ile Lieu. Phe Luell Phe Lell Met Ser Luell Ala 115 12 O 125

Gly Ile Luell Asn His Lieu. Ile Phe Phe Phe Gly Ser Asp Phe Glu 13 O 135 14 O

Asn Tyr Ile Arg Thir Ile Thir Thr Thir Ile Ser Pro Lell Luell Luell Ile 145 150 155 160

Pro

<210s, SEQ ID NO 43 &211s LENGTH: 18O 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 43

Met Ser Glu Ser Glu Lell Gln Lieu. Wall Ala Arg Arg Ile Arg Ser Phe 1. 5 15

Pro Asp Phe Pro Ile Pro Gly Val Luell Phe Arg Asp Ile Ser Pro Luell 25

Lell Asp Pro Asp Ser Phe Arg Ala Ser Ile Arg Lell Luell Ala Gly 35 4 O 45

His Luell Ser Thir His Gly Gly Ile Asp Tyr Ile Ala Gly Luell SO 55 6 O

Asp Ser Gly Phe Lell Phe Gly Pro Ser Luell Ala Glin Glu Luell Gly 65 70

Wall Gly Wall Lell Ile Arg Llys Arg Gly Lys Lell Pro Gly Pro Thir 85 90 95

Wall Ser Ala Ser Tyr Ser Lieu. Glu Tyr Gly Lys Ala Glu Luell Glu Ile 105 11 O

Glin Asp Ala Lell Glu Pro Gly Glin Wall Wall Ile Wall Asp Asp 115 12 O 125

Lell Luell Ala Thir Gly Gly Thir Met Ala Ala Cys Glu Luell Luell Ser 13 O 135 14 O

Glin Luell Arg Ala Glu Wall Wall Glu Wall Ser Lell Wall Glu Luell Thir 145 150 155 160

Ser Luell Gly Arg Glu Llys Lieu. Gly Pro Wall Pro Phe Phe Ser Luell 1.65 17O 17s

Lell Glin Glu 18O

SEQ ID NO 44 LENGTH: 22O TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 44 Met Gly Ser Lys Lieu. Ser Llys Llys Llys Lys Gly Tyr Asn. Wall Asn Asp 1. 5 15 Glu Lys Ala Lys Asp Lys Asp Llys Lys Ala Glu Gly Ala Gly Thr Glu 25 US 7,972,785 B2 167 168 - Continued

Glu Glu Gly Thir Glin Glu Ser Glu Pro Glin Ala Ala Ala Asp Ala 35 4 O 45

Thir Glu Wall Glu Ser Ala Glu Glu Pro Lys Asp Ala Asp SO 55 6 O

Gly Glu Ala Ala Glu Glu Glu Ala Asp Ala Ala Lys 65 70

Glu Glu Ala Pro Lys Ala Glu Pro Glu Lys Ser Gly Ala Glu 85 90 95

Glu Glin Pro Glu Pro Ala Pro Ala Pro Glu Glin Ala Ala Pro 105

Gly Pro Ala Ala Gly Gly Glu Ala Pro Ala Glu Ser Ala 115 12 O 125

Glu Ser Thir Gly Ala Ala Asp Gly Ala Pro Glin Glu Glu Ala 13 O 135

Lys Thir Glu Ala Pro Ala Ala Gly Pro Glu Ser Asp Ala 145 150 155 160

Ala Pro Ala Ala Ser Asp Ser Pro Ser Thir Pro Ala Pro Ser 1.65 17O 17s

Ser Glu Thir Pro Ala Ala Ser Glu Ala Pro Ser Ser Ala Ala 18O 185 19 O

Ala Pro Ala Pro Ala Ala Pro Ala Ala Glu Pro Glin Ala Glu Ala Pro 195 2OO 2O5

Wall Ala Ser Ser Glu Glin Ser Wall Ala Wall Glu 21 O 215 22O

<210s, SEQ ID NO 45 &211s LENGTH: 169 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 45

Met Ser Glin Ser Trp Wall Pro Ala Wall Gly Luell Thir Lell Wall Pro Ser 1. 5 15

Lell Gly Gly Phe Met Gly Ala Phe Wall Arg Gly Glu Gly Luell Arg 25

Trp Ala Ser Lell Glin Pro Ser Trp His Pro Pro Arg Trp Thir 35 4 O 45

Lell Ala Pro Ile Trp Gly Thir Luell Tyr Ser Ala Met Gly Tyr Gly Ser SO 55 6 O

Tyr Ile Ile Trp Glu Lell Gly Gly Phe Thir Glu Glu Ala Met Wall 65 70

Pro Luell Gly Luell Tyr Thir Gly Glin Luell Ala Luell Asn Trp Ala Trp Pro 85 90 95

Pro Ile Phe Phe Gly Ala Arg Glin Met Gly Trp Ala Lell Wall Asp Luell 105 11 O

Met Luell Wall Ser Gly Wall Ala Thir Ala Thir Thir Lell Ala Trp His Arg 115 12 O 125

Wall Ser Pro Pro Ala Ala Arg Luell Luell Tyr Pro Tyr Lell Ala Trp Luell 13 O 135 14 O

Ala Phe Ala Thir Met Lell Asn Wall Trp Arg Asp Asn Ser Gly 145 150 155 160

Arg Arg Gly Gly Ser Arg Lell Thir Glu 1.65

<210s, SEQ ID NO 46 &211s LENGTH: 277 US 7,972,785 B2 169 170 - Continued

212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 46 Met Ser Ser Asp Arg Pro Wall Ala Lieu Val Thr Gly Ala Asn Lys Gly 1. 5 1O 15 Ile Gly Phe Ala Ile Val Arg Asp Lieu. Cys Arg Llys Phe Lieu. Gly Asp 2O 25 3O Val Val Lieu. Thir Ala Arg Asp Glu Ser Arg Gly His Glu Ala Val 35 4 O 45

Glin Lieu. Glin Thr Glu Gly Lieu. Ser Pro Arg Phe His Glin Lieu. Asp Ile SO 55 6 O

Asp ASn Pro Glin Ser Ile Arg Ala Lieu. Arg Asp Phe Lieu. Lieu. Glin Glu 65 70 7s 8O Tyr Gly Gly Lieu. Asn Val Lieu Val Asn. Asn Ala Gly Ile Ala Phe 85 90 95 Val Val Asp Pro Thr Pro Phe His Ile Glin Ala Glu Val Thr Met 1OO 105 11 O

Thr Asn Phe Phe Gly Thr Glin Asp Val Cys Lys Glu Lieu. Leu Pro Ile 115 12 O 125

Ile Llys Pro Glin Gly Arg Val Val Asn Val Ser Ser Ser Val Ser Luell 13 O 135 14 O

Arg Ala Lieu Lys Ser Cys Ser Pro Glu Lieu. Glin Gln Llys Phe Arg Ser 145 150 155 160

Glu Thir Ile Thr Glu Glu Glu Lieu Val Gly Lieu Met Asn Llys Phe Ile 1.65 17O 17s

Glu Asp Ala Lys Lys Gly Val His Ala Lys Glu Gly Trp Pro Asn Ser 18O 185 19 O Ala Tyr Gly Val Thr Lys Ile Gly Val Thr Val Lieu Ser Arg Ile 195 2OO 2O5

Ala Arg Llys Lieu. Asn. Glu Glu Arg Arg Glu Asp Llys Ile Lieu. Lieu. Asn 21 O 215 22O

Ala Cys Cys Pro Gly Trp Val Arg Thr Asp Met Ala Gly Pro Llys Ala 225 23 O 235 24 O

Thr Lys Ser Pro Glu Glu Gly Ala Glu Thr Pro Val Tyr Lieu. Ala Luell 245 250 255 Lieu Pro Pro Gly Ala Glu Gly Pro His Gly Glin Phe Val Glin Asp 26 O 265 27 O Llys Val Glu Pro Trp 27s

<210s, SEQ ID NO 47 &211s LENGTH: 3O3 212. TYPE: PRT <213> ORGANISM: Rattus sp. <4 OOs, SEQUENCE: 47 Met Trp Pro Lieu Ala Ala Ala Lieu. Lieu. Lieu. Gly Ser Cys Cys Cys Gly 1. 5 1O 15

Ser Ala Glin Lieu Lleu Lleu Ser Llys Val Lys Ser Val Glu Phe Thr Ser 2O 25 3O

Cys Asn Asp Thr Val Val Ile Pro Cys Llys Val Lieu. Asn Val Glu Ala 35 4 O 45 Glin Ser Thr Asp Glu Met Phe Val Llys Trp Llys Lieu. Asn Llys Ser Tyr SO 55 6 O

Ile Phe Ile Tyr Asp Gly Asn Lys Asn Ser Thr Thr Arg Glu Gln Asn US 7,972,785 B2 171 172 - Continued

65 70

Phe Thir Ser Ala Lys Ile Ser Wall Ser Asp Luell Lell Lys Gly Ile Ala 85 90 95

Ser Luell Thir Met Asp Thir His Glu Ala Wall Wall Gly Asn Tyr Thir 105 11 O

Glu Wall Thir Glu Lell Ser Arg Glu Gly Thir Wall Ile Glu Luell 115 12 O 125

Asn Arg Pro Wall Ser Trp Phe Ser Thir Asn Glu Lys Ile Luell Ile Wall 13 O 135 14 O

Ile Phe Pro Ile Lell Ala Ile Luell Luell Phe Trp Gly Phe Gly Ile 145 150 155 160

Lell Thir Luell Tyr Ser Ser His Thir ASn Arg Ile Ile Luell 1.65 17s

Lell Luell Wall Ala Gly Lell Ala Luell Thir Luell Ile Wall Wall Wall Gly Ala 18O 185 19 O

Ile Luell Phe Ile Pro Gly Glu Lys Pro Wall Asn Ala Ser Gly Luell 195

Gly Luell Ile Wall Ile Ser Thir Gly Ile Luell Ile Lell Lell Glin Tyr Asn 21 O 215

Wall Phe Met Thir Ala Phe Gly Met Thir Ser Phe Thir Ile Ala Ile Luell 225 23 O 235 24 O

Ile Thir Glin Wall Lell Gly Wall Luell Ala Wall Wall Gly Met Cys Luell 245 250 255

Ile Met Ala Cys Glu Pro Wall His Gly Pro Lell Lell Ile Ser Gly 26 O 265 27 O

Lell Gly Ile Ile Ala Lell Ala Glu Luell Luell Gly Lell Wall Met 28O 285

Phe Wall Ala Ser Asn Glin Arg Thir Ile Glin Pro Pro Arg Asn Asn 29 O 295 3 OO

<210s, SEQ ID NO 48 &211s LENGTH: 328 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 48

Met Arg Ser His Thir Gly Lell Arg Ala Luell Wall Ala Pro Gly Cys Ser 1. 5 15

Lell Luell Luell Luell Tyr Lell Lell Ala Ala Thir Arg Pro Asp Arg Ala Wall 2O 25

Gly Asp Pro Ala Asp Ser Ala Phe Thir Ser Luell Pro Wall Arg Glu Glu 35 4 O 45

Met Met Ala Tyr Ala Asn Luell Ser Luell Glu Thir Asn Ile Ser SO 55 6 O

Lell Thir Glu Glin Thir Arg Wall Ser Glu Glin ASn Ile Thir Luell Glu Arg 65 70

Pro Ser His Luell Glu Lell Glu Thir Phe Thir Ala Thir Glu Asp Wall 85 90 95

Met Ser Met Asn Wall Thir Trp Lys Asp Asp Ala Lell Luell Glu Thir 105 11 O

Thir Asp Gly Phe Asn Thir Thir Lys Met Gly Asp Thir Lell Tyr Ser Glin 115 12 O 125

Arg Phe Thir Wall Phe Asn Ser Glin Met Gly Lys Ser 13 O 135 14 O

Phe Luell Gly Glu Glu Lell Arg Gly Thir Phe ASn Ile Arg Wall Pro US 7,972,785 B2 173 174 - Continued

145 150 155 160

Wall His Gly Asn Pro Luell Ile Thir Wall Gly Asp Ser Thir 1.65 17s

Wall Luell Cys Glu Glin Asn Cys Luell Pro Lell Asn Trp Thir Trp 185 19 O

Met Ser Asn Gly Thir Ala Glin Wall Pro Ile Asp Wall His Wall Asn 195

Asp Lys Phe Asp Ile Asn Gly Ser Ala ASn Glu Thir Luell Lys 21 O 215 22O

Wall His Luell Lell Glu Glu Asp Gly Gly Ser Trp Arg Ala 225 23 O 235 24 O

Ala Phe Pro Luell Gly Glu Ser Glu Glu His Ile Lell Wall Wall Luell 245 250 255

Ser Phe Met Wall Pro Lell Pro Phe Luell Ala Ile Ile Ala Glu Wall 26 O 265 27 O

Ile Lel Luell Wall Ala Ile Ile Luell Luell Glu Wall Tyr Thir Glin Lys 285

Lys Asn Asp Pro Asp Asp Gly Glu Phe Glu Glin Ile Glu Glin 29 O 295 3 OO

Lell Ser Asp Asp Ser Asn Gly Ile Glu ASn Asn Wall Pro Arg Tyr 3. OS 310 315 32O

Arg Thir Asp Ser Gly Asp Glin 3.25

<210s, SEQ ID NO 49 &211s LENGTH: 212. TYPE : PRT <213> ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 49

Met Ala Pro Ser Pro Arg Pro Glin His Wall Luell His Trp Glu Ala 1. 5 15

His Ser Phe Tyr Lell Lell Ser Pro Luell Met Gly Phe Lell Ser Arg Ala 2O 25

Trp Ser Arg Luell Arg Gly Pro Glu Wall Ser Glu Ala Trp Luell Ala Glu 35 4 O 45

Thir Wall Ala Gly Ala Asn Glin Ile Glu Ala Asp Ala Lell Luell Thir Pro SO 55 6 O

Pro Pro Wall Ser Glu Asn His Luell Pro Luell Arg Glu Thir Glu Gly Asn 65 70

Thir Pro Glu Trp Ser Ala Ala Glin Arg Lell Luell Asp Wall 85 90 95

Ala Glin Ser Ser Pro Pro Thir Trp Gly Lell Ser Asp Ile Asp 105 11 O

His Asn Gly Lys Pro Gly Glin Asp Gly Luell Arg Glu Glin Glu Wall 115 12 O 125

His Thir Ala Gly Lell Pro Thir Luell Glin Pro Lell His Luell Glin Gly 13 O 135 14 O

Asp Wall Gly Glu Wall Wall Ala Arg Glu Glu Gly Wall Ser 150 155 160

Luell Ala Pro Thir Ser His Trp Glu Gly Gly Pro Ala Glu Asp 1.65 17O

Glu Asp Thir Glu Thir Wall Lys Ala His Glin Ala Ser Ala Ala 18O 185 19 O

Ser Ile Ala Pro Gly Pro Ser Thir Ser Wall Pro Gly US 7,972,785 B2 175 176 - Continued

195 2OO 2O5

Glu Ala Glu His Arg Ala Thir Glu Glu Lys Gly Thir Asp Asn Ala 21 O 215 22O

Glu Pro Ser Gly Ser His Ser Arg Wall Trp Glu His Thir Arg Glu 225 23 O 235 24 O

Arg Pro Glin Glu Gly Glu Thir Pro Glu Glin His Arg Ala Gly 245 250 255

Glin Ser His Pro Cys Glin Asn Ala Glu Ala Glu Glu Gly Gly Pro Glu 26 O 265 27 O

Thir Ser Wall Ser Gly Ser Ala Phe Luell Lys Ala Trp Wall Arg 285

Pro Gly Glu Asp Thir Glu Glu Glu Glu Asp Ser Asp Lell Asp Ser Ala 29 O 295 3 OO

Glu Glu Asp Thir Ala His Thir Thir Thir Pro His Thir Ser Ala Phe 3. OS 310 315

Lell Ala Trp Wall Arg Pro Gly Glu Asp Thir Glu Glu Glu Asp 3.25 330 335

Asp Gly Asp Trp Asp Ser Ala Glu Glu Asp Thir Ala Glin Ser Thir 34 O 345 35. O

Thir Pro His Thir Ser Ala Phe Luell Ala Trp Wall Tyr Arg Pro Gly 355 360 365

Glu Asp Thir Glu Glu Glu Asp Asp Ser Glu ASn Wall Ala Pro Wall Asp 37 O 375

Ser Glu Thir Wall Asp Ser Glin Ser Thir Glin His Luell Pro Wall 385 390 395 4 OO

Glu Thir Gly Gly Glu Ala Glu Pro Pro Pro Phe Glin Wall 4 OS 415

Ala Phe Luell Pro Gly Glin Pro Ala Pro Pro Trp Ala Ala Pro 42O 425 43 O

Luell Pro Luell Arg Lell Glin Lys Arg Luell Arg Ser Phe Ala Pro 435 44 O 445

Ala Arg Asn Glin Asp Pro Glu Ile Pro Luell Lys Gly Arg Wall His 450 45.5 460

Phe Ser Glu Wall Thir Wall His Phe Luell Ala Wall Trp Ala Gly Pro 465 470

Ala Glin Ala Ala Arg Arg Gly Pro Trp Glu Glin Phe Ala Arg Asp Arg 485 490 495

Ser Arg Phe Ala Arg Arg Ile Ala Glin Ala Glu Glu Glin Luell Gly Pro SOO 505

Luell Thir Pro Ala Phe Arg Ala Arg Ala Trp Thir Arg Luell Arg Asn 515 525

Lell Pro Luell Pro Lell Ser Ser Ser Ser Luell Pro Lell Pro Glu Pro 53 O 535 54 O

Ser Ser Thir Glu Ala Thir Pro Luell Ser Glin Asp Wall Thir Thir Pro Ser 5.45 550 555 560

Pro Luell Pro Ser Glu Ile Pro Pro Pro Ser Luell Asp Lell Gly Gly Arg 565 st O sts Arg Gly

SEO ID NO 5 O LENGTH: 357 TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: SO US 7,972,785 B2 177 178 - Continued

Met Ala Glu Luell Glin Glu Wall Glin Ile Thir Glu Glu Lys Pro Luell Luell 15

Pro Gly Glin Thir Pro Glu Ala Ala Lys Thir His Ser Wall Glu Thir Pro 25 3O

Gly Ser Wall Thir Phe Thir Wall Gly Thir Pro Lys Pro 35 4 O 45

Pro Ala Ile Phe Thir His Asp Wall Gly Luell Asn Ser Cys SO 55 6 O

Phe Glin Pro Luell Phe Glin Phe Gly Asp Met Glin Glu Ile Ile Glin Asn 65 70

Phe Wall Arg Wall His Wall Asp Ala Pro Gly Met Glu Glu Gly Ala Pro 85 90 95

Wall Phe Pro Luell Gly Glin Tyr Pro Ser Glin Asp Glin Luell Ala Asp 1OO 105 11 O

Met Ile Pro Ile Lell Glin Tyr Luell Asn Phe Ser Thir Ile Ile Gly 115 12 O 125

Wall Gly Wall Gly Ala Gly Ala Tyr Ile Lel Ser Arg Ala Luell Asn 13 O 135 14 O

His Pro Asp Thir Wall Glu Gly Luell Wall Lel Ile Asn Ile Asp Pro Asn 145 150 155 160

Ala Gly Trp Met Asp Trp Ala Ala His Lell Thir Gly Luell Thir 1.65 17s

Ser Ser Ile Pro Glu Met Ile Luell Gly His Luell Phe Ser Glin Glu Glu 18O 185 19 O

Lell Ser Gly Asn Ser Glu Lell Ile Glin Arg Ser Luell Ile Thir 195

His Ala Pro Asn Lell Glu Asn Ile Glu Lel Trp Asn Ser Asn 21 O 215

Asn Arg Arg Asp Lell Asn Phe Glu Arg Gly Gly Glu Met Thir Luell Lys 225 23 O 235 24 O

Pro Wall Met Lell Wall Wall Gly Asp Glin Ala Pro His Glu Asp Ala 245 250 255

Wall Wall Glu Cys Asn Ser Luell Asp Pro Thir Glin Thir Ser Phe Luell 26 O 265 27 O

Met Ala Asp Ser Gly Gly Glin Pro Glin Luell Thir Glin Pro Gly Lys 285

Lell Thir Glu Ala Phe Tyr Phe Wall Glin Gly Met Gly Met Ala 29 O 295 3 OO

Ser Ser Met Thir Arg Lell Ser Arg Ser Arg Thir Ala Ser Luell Thir 3. OS 310 315

Ser Ala Ala Ser Ile Asp Gly Ser Arg Ser Arg Ser Arg Thir Luell Ser 3.25 330 335

Glin Ser Ser Glu Ser Gly Thir Luell Pro Ser Gly Pro Pro Gly His Thir 34 O 345 35. O

Met Glu Wall Ser Cys 355

SEO ID NO 51 LENGTH: 152 TYPE : PRT ORGANISM: Rattus sp.

< 4 OOs SEQUENCE: 51 Met Ala Asn. Ser Glu Arg Thr Phe Ile Ala Ile Llys Pro Asp Gly Val 1. 5 15 US 7,972,785 B2 179 180 - Continued

Glin Arg Gly Luell Wall Gly Glu Ile Ile Arg Phe Glu Glin Lys Gly 2O 25

Phe Arg Luell Wall Gly Lell Lys Phe Ile Glin Ala Ser Glu Asp Luell Luell 35 4 O 45

Glu His Ile Asp Lell Asp Arg Pro Phe Phe Ser Gly Luell SO 55 6 O

Wall Met His Ser Gly Pro Wall Wall Ala Met Wall Trp Glu Gly 65 70

Lell Asn Wall Wall Lys Thir Gly Arg Wall Met Luell Gly Glu Thir Asn Pro 85 90 95

Ala Asp Ser Lys Pro Gly Thir Ile Arg Gly Asp Phe Ile Glin Wall 105 11 O

Gly Arg Asn Ile Ile His Gly Ser Asp Ser Wall Glu Ser Ala Glu 115 12 O 125

Glu Ile Ser Luell Trp Phe Glin Pro Glu Glu Luell Wall Asp Ser 13 O 135 14 O

Cys Ala Glin Asn Trp Ile Glu 145 150

<210s, SEQ ID NO 52 &211s LENGTH: 636 212. TYPE : PRT &213s ORGANISM: Rattus sp.

<4 OOs, SEQUENCE: 52 Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Gly Lys 1. 5 15

Asp Arg Gly Ser Arg Gly Pro Gly Pro Ala Glu Gly Asp Pro Ser 2O 25 3O

Pro Ala Luell Pro Pro Arg Lell Lys Glu Met Lys Ser Glin Glu Ser Ala 35 4 O 45

Ala Gly Ser Lell Wall Lell Arg Glu Thir Ser Ser Glu Tyr Ser SO 55 6 O

Ser Luell Arg Phe Trp Phe Asn Gly ASn Glu Lell Asn Arg Lys 65 70

Asn Pro Glu Ile Ile Glin Lys Lys Pro Gly Ser Glu 90 95

Lell Arg Ile Asn Ala Ser Luell Ala Asp Ser Gly Glu Tyr Met 105 11 O

Wall Ile Ser Lell Gly Asn Asp Ser Ala Ser Ala Asn Ile Thir 115 12 O 125

Ile Wall Glu Ser Asn Glu Phe Ile Thir Gly Met Pro Ala Ser Thir Glu 13 O 135 14 O

Thir Ala Wall Ser Ser Glu Ser Pro Ile Arg Ile Ser Wall Ser Thir 145 150 155 160

Glu Gly Ala Asn Thir Ser Ser Ser Thir Ser Thir Ser Thir Thir Gly Thir 1.65 17O 17s

Ser His Luell Ile Lys Ala Glu Lys Glu Lys Thir Phe Cys Wall Asn 18O 185 19 O

Gly Gly Glu Phe Thir Wall Lys Asp Luell Ser Asn Pro Ser Arg 195

Lell Cys Pro Asn Glu Phe Thir Gly Asp Arg Glin Asn Tyr 21 O 215

Wall Met Ala Ser Phe Tyr Ala Glu Glu Luell Tyr Glin Arg Wall 225 23 O 235 24 O