bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1 TITLE: PCV2 aggravated SS2 infection directly showed from differentially 2 expressed proteome of PK15 cells.
3 Tan Jimin1,2, Xiao Qi2,3,4, Li Xingguang5, Fan Hongjie1*, He Kongwang2,3,4*
4
5
6 1College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China
7 2Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation
8 Base Ministry of Science and Technology, Nanjing, China
9 3Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences-Key
10 Laboratory of Veterinary Biological Engineering and Technology, Ministry of
11 Agriculture and Rural Affairs, Nanjing, China
12 4Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal
13 Infectious Disease and Zoonose, Yangzhou University, Yangzhou, China
14 5Hubei Engineering Research Center of Viral Vector, Wuhan University of
15 Bioengineering, Wuhan 430415, China.
16 Correspondence:
17 Fan Hongjie ([email protected])
18 He Kongwang ([email protected])
19
20
21 Running title: PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15
22
23 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 24 Abbreviations:
25 CCK8: Cell counting kit-8
26 CY3: Cyanine3 carboxylic acid
27 ECM: Extracellular matrix
28 FBS: Fetal bovine serum saline
29 FITC: Fluorescein isothiocyanate
30 HPLC: High performance liquid chromatography
31 IDA: Information Dependent Acquisition
32 IPA: Ingenuity Pathways Analysis
33 IP-MS: Immunoprecipitation-Mass Spectrometry
34 iTRAQ: Isobaric tags trace quantification
35 KEGG: Kyoto Encyclopedia of Genes and Genomes
36 LC-ESI-MS/MS: Liquid chromatography-electrospray tandem mass spectrometry
37 MOI: Multiplicity of infection
38 MSI: Mass spectrometry image
39 PBS: Phosphate buffer saline
40 PCAD: Porcine Circovirus Associated Disease
41 PCV2: Porcine circovirus type 2
42 PDNS: Swine Dermatitis and Nephrotic Syndrome
43 PK15: Porcine Kidney cell 15
44 PWMS: Post-weaning multiple system failure syndrome
45 SILAC: The stable isotope labeling of amino acids in cell culture
46 SS2: Streptococcus suis 2
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 47 STRING: Search Tool for the Retrieval of Interacting Genes/Proteins
48 THB: Todd Hewitt Broth
49 TMT: tandem mass tags
50 TEAB: triethanolamine buffered
51
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15
52 ABSTRACT
53 PCV2 and SS2 were clinically two major pathogens in pigs and they were zoonotic
54 pathogens. There was extensive cellular tropism for both PCV2 and SS2, as well as,
55 PK15 cells was PCV2 infection mainly cell model. It was found that when PCV2 infected
56 PK15 cells before at the MOI=0.1, SS2 could cause more damage to PK15 cells. ITRAQ
57 labeling proteomic technology was used to explore the differentially expressed proteome
58 of PCV2 and SS2 single infection and co-infection in PK15 cells. The results showed that
59 there were total 4736 proteins distinct changed in this infection models for PK15 cells.
60 PCV2 aggravated SS2 infection were showed directly in the big amount of differentially
61 expressed proteins like AGO3, OSBPL1A, ALB, RIC8A, UBL4A, INTS5 and so on. For
62 the KEGG pathway analysis, it indicated that PCV2 mainly induced the proteins lessen
63 though a series of disease pathway like metabolic pathways, huntington’s disease, insulin
64 signaling pathways, long-term depression, etc. to make cell at a state of compensation.
65 PCV2 before infection made the cells was on the chopping block. Contemporary, SS2
66 induced the proteins of PK15 rapidly changing, including pathways like spliceosome,
67 endoplasmic reticulum, tight junction, actin cytoskeleton and some involved in parasitic
68 infections pathways like ECM-receptor interaction, Leishmaniasis, Toxoplasmosis and so
69 on. Simultaneously, PCV2 and SS2 existed common ground that they influence PK15
70 cell by some virulence factor interacted with cytomembrane, influenced the function of
71 ribosome and tRNA. The role of SS2 in the co-infection like a cook chopper.
72 KEYWORDS: PCV2, SS2, iTRAQ, co-infection, proteomics, pathways
73
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 74 INTRODUCTION
75
76 Porcine circovirus type 2 (PCV2) was a single stranded, non-capsular, closed circular
77 DNA virus including 1,766-1,768 nucleotides 1-3. PCV2 was pivotal pathogen, involving
78 in porcine circovirus related disease, porcine dermatitis and nephropathy syndrome,
79 PCV2 reproductive disease, PCV2 lung disease, severe systemic PCV2 infection and
80 PCV2 enteric disease 4-12. Porcine circovirus-related diseases were considered to be
81 multifactorially affected diseases in the swine industry 13, of which can promote
82 co-infection with other viruses or bacteria 14-16, causing aggravation or sharpness of the
83 disease 4. PCV2 can damage the pig's immune system, characterized by
84 lymphadenopathy, lymphopenia, apoptosis and autophagy, leading to severe
85 immunosuppression, causing huge chance to the other agent infection 17-20. At the same
86 time, PCV2 could lead to zoonotic disease 21,22 and kept persistently infection in PK15
87 cells 23. Streptococcus suis (S. suis) was a Gram-positive facultative anaerobic bacterium,
88 of which type 2 was most serious pathogen of pigs and could be transmitted to humans
89 through close contact with pigs 24,25. It was the cause of porcine polyserositis and other
90 diseases, such as heart inflammation, brain infection, bronchial inflammation, arthritis
91 syndrome, sepsis and abortion etc. 26-30. According to the clinical detection data, the
92 infection rates of the two pathogens in the healthy and sick pigs were 17% and 35%,
93 respectively, accounting for 69.5% and 59.4% of the total infection or co-infection of the
94 two pathogens, indicating that co-infection of the two pathogens was more common in
95 the clinic in China 31. The co-infection of the two pathogens was also reported in Korea,
96 but the symptom was atypical like PCAD 32. When pathogens infected host cells, the
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 97 progression of infection depended on host-pathogen interaction that were regulated in
98 time and space, nevertheless, this response was complex, and the most intuitive
99 performance was the differential proteins changed in host cells 33,34. These interactions
100 represent anti-pathogenic and pro-pathogenic cellular responses. So, proteomics study
101 about the host-pathogen involves many aspects. First, we should to explore the
102 differential proteins between treatment and control groups. As the progress of protein
103 labeling and quantification techniques combine with Mass spectrometry (MS), like,
104 label-free MS quantification, which was easy, multifunctional, and could be applied to
105 any biological system. Successively, the stable isotope labeling of amino acids in cell
106 culture (SILAC) 35, tandem mass tags (TMT) 36 or other isobaric tags trace quantification
107 (iTRAQ) 36 were emerge and mature applied in pathogen and host. In studying host-virus
108 interactions, SILAC was employed to study hepatitis C virus 37. Proteomics study of
109 porcine circovirus types 2 and 3 on macrophages and porcine lung tissue has been
110 reported 38,39. For bacteria, IP-MS was used to identify interactions between effector
111 proteins secreted by intracellular Salmonella and host proteins, and SILAC quantification
112 helped assess specificity of interactions 40 and ITRAQ was used to vaccine selection for
113 Mycoplasma bovis 41. At present, vaccines were mainly used to prevent and treat viral
114 diseases and antibiotics were used to treat bacterial diseases. However, there was still a
115 lack of drugs for the treatment of viral diseases, meanwhile, the mutation rate of the virus
116 was very quickly, and bacterial resistance was also a serious problem, bacterial disease
117 treatment was still a big challenge. As organism was always exposed to bacteria and virus
118 environment, the changes of the organism should be studied to understand the pathogenic
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 119 mechanism of viruses and bacteria and to provide theoretical guidance for the prevention
120 and treatment 42.
121
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 122 MATERIALS AND METHODS
123
124 Cells, viruses, bacteria and antibodies
5.75 125 The PCV-free porcine kidney epithelial cell lines (PK15) and PCV2 (10 TCID50 /ml)
126 strain, which frozen in liquid nitrogen at -80℃, respectively, were kept in Jiangsu
127 Academy of Agricultural Sciences -Institute of Veterinary Medicine（JAAS-IVM）
128 laboratory and maintained in Dulbecco's Modified Eagle Media (DMEM; Gibco,
129 Carlsbad, CA) containing 7% gamma-irradiated fetal bovine serum (FBS, Gibco). SS2-1
130 stored in 50% glycerol was kept in JAAS-IVM. Bacterial cultured in Todd Hewitt Broth
131 (THB) medium at 37℃ 200 r/min. The polyclonal antibody (pAb) and monoclonal
132 antibody (mAb) against the PCV2 Cap protein and SS2 were obtained as previously and
133 the working concentration was used in 1:1000 and 1:400 dilution, respectively. The
134 polyclonal antibody DExH-box helicase 9 (DHX9) (ABclonal), RIC8A guanine
135 nucleotide exchange factor A (RIC8A) (ABclonal), Annexin 4 (ANXA4) (ABclonal),
136 L-lactate dehydrogenase (LDHA) (ABclonal) were used in 1:1000 dilution, and mAb
137 proliferating cell nuclear antigen (PCNA) (abcam) and glyceraldehyde-3-phosphate
138 dehydrogenase (GAPDH) (ABclonal) were used in 1:10000 dilution.
139
140 Cell culture, virus and bacterial inoculation
141 There were 8 groups. The 1st and 2nd groups were the blank cells with no deal with for
142 cultured with 50h and 54h named Blank-2h and Blank-6h. The 3rd and 4th groups were
143 the blank cells infected with PCV2 (MOI=0.1) cultured with 50h and 54h named
144 PCV2-2h and PCV2-6h. The 5th and 6th groups were the blank cells cultured with 48h
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 145 and then infected with SS2 (MOI=10) with 2h and 6h named SS2-2h and SS2-6h. The
146 7th and 8th groups were the blank cells infected with PCV2 (MOI=0.1) for 48h and then
147 infected with SS2 (MOI=10) for 2h and 6h named PCV2-SS2-2h and PCV2-SS2-6h.
148 PK15 cells were passaged to 3rd generation and maintained at 37°C with 5% CO2 for
149 different use. After 48 hours, the culture conditions of the cells were in DMEM with 7%
150 FBS and the virus was in DMEM with 2% FBS.
151
152 The cell viability assay
153 PK15 cells inoculated with PCV2 (MOI=0.1) was prepared for 48h earlier, the same as
154 blank PK15 cells. PK15 cells was infected with different dose (MOI=0.1, 1, 2.5, 5 and 10)
155 and different co-culture times (2h, 4h, 6h, 12h, and 16h) for SS2. Then CCK8 solution
156 was added 10µL in each well. In order to permitted the formazan dye adhere to the alive
157 cell and reactive, each plate was placed at 37 °C for 2h. For detection, the cell plate was
158 shaken for 10min. Then, a microplate reader (Bio-Rad, model 450) was used to measure
159 its absorbance of each well. Cell viability assay at the formula：
160
! "#$%_''% -!)("#$_''%) 161 %Viability= ×100% ! ,-./0 -!)(,-./0)
162
! ""# -!%(""#) 163 %Viability= ×100% ! ()*+, -!%(()*+,)
164
165 A(PCV2_SS2): the absorbance of PK15 cell co-infected with PCV2-SS2
166 A0(PCV2_SS2): the absorbance of the only cell culture fluid co-infected with PCV2-SS2
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 167 A(SS2): the absorbance of PK15 cell co-infected with SS2
168 A0(PCV2_SS2): the absorbance of the only cell culture fluid co-infected with SS2
169 A(Blank): the absorbance of PK15 cell without pathogen infection
170 A0(Blank): the absorbance of the only cell culture fluid
171
172 Determination of iTRAQ experimental protocol
173 The different group samples were prepared according to experimental design mentioned
174 in preceding part of this text. In order to achieve the comparison and inspections of
175 different groups. The overall design of proteomics was shown in Figure 2. A certain
176 quantity sample was taken from each group of the 8 groups in a ratio of 1:1…1:1 for
177 mixing and forming a new group. The 9 groups were marked separately and reclassified
178 into two new groups for iTRAQ assay according to the time 2 hours and 6 hours. This
179 was done to make it easier to compare changes between groups.
180
181 Protein preparation
182 Different group samples were added with protein lysate (7M Urea, 2M Thiourea, 4%
183 SDS, 40mM Tris-HCl pH8.5, 1mM PMSF, 2mM EDTA) mixing and 5 minutes in ice,
184 then, added a final concentration of 10mM DTT in ultrasound and ice for 15min. The
185 treated samples were centrifuged at 13000g, 4°C for 20 minutes. The different
186 supernatants were picked in new centrifuge tubes, added cold acetone overnight at -20°C,
187 then centrifuged at 13000g, 4°C for 20 minutes and collecting sediment and dried in air.
188 The treated samples dissolved in 8M Urea, 100mM TEAB (pH8.0) and added DTT to a
189 final concentration of 10mM, water bath in 56°C for 30 minutes. Subsequently, the
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 190 samples added IAM to a final concentration of 55 mM putting in dark place at room
191 temperature for 30 min. Bradford method was used to measure of protein concentration.
192
193 Digestion and desalting
194 The same amount of protein from each sample was used for trypsin digestion. For each
195 sample, 100µg of protein was diluted 5 times, and then added trypsin at a mass ratio of
196 1:50 (trypsin: protein), digesting overnight at 37°C. The samples after enzymatic
197 hydrolysis was desalted on a C18 column, and the dehydrated peptide was freeze-dried
198 under vacuum.
199
200 iTRAQ labeling and fractionation
201 The dried peptide was solubilized with 0.5M 20uL TEAB for peptides labeling. Samples
202 were labeled with iTRAQ Reagent-8 plex Multiplex Kit (AB Sciex U.K. Limited)
203 according to the manufacturer instructions. All of the labeled samples were mixed with
204 equal amount. Then, the labeled samples were fractionated using high performance liquid
205 chromatography (HPLC) system (Thermo DINOEX Ultimate 3000 BioRS) using a
206 Durashell C18(5 um, 100Å, 4.6 × 250 mm). Peptides separation was achieved using a
207 gradually increasing ACN concentration under alkaline conditions at a flow rate of 1
208 ml/min and one tube was collected per minute. A total of 42 secondary fractions were
209 collected and combined into 12 fractions. The combined fractions were desalted on a
210 Strata-X column and dried under vacuum.
211
212 Proteomics LC-MS/MS Analysis
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 213 LC-ESI-MS/MS analysis was performed on an AB SCIEX nanoLC-MS/MS (Triple TOF
214 5600 plus) system. Samples were chromatographed using a 90min gradient from 2–98%
215 (buffer A 2% (v/v) acetonitrile and 0.1% (v/v) formic acid, buffer B 98% (v/v)
216 acetonitrile, and 0.1% (v/v) formic acid) after direct injection onto a 20µm PicoFrit
217 emitter (New Objective) packed to 12cm with Strata-X C18 AQ 3µm 120 Å stationary
218 phase. For IDA (Information Dependent Acquisition), MSI was performed with
219 fragmentation accumulation time of 250ms, and MS2 collected 30 precursor ions with
220 fragmention accumulation time of 50ms. MS1 and MS2 collected the range 350-1500m/z
221 and the range of 100-1500m/z, respectively. The precursors fragmentation was excluded
222 from reselection for the first 15s.
223
224 Proteomics Data Analysis
225 ProteinPilotTM Software V4.5 was used to acquisition the original data file from MS/MS
226 directly. The Paragon algorithm2 was integrated into ProteinPilot and employed against
227 Uniprot Sus scrofa species (PR1-18110005 items, update in Nov. 2018) for database
228 searching and for protein identification. The parameters were set as follows: the
229 instrument was TripleTOF 5600, iTRAQ quantification, cysteine modified with
230 iodoacetamide; biological modifications were selected as ID focus, trypsin digestion, the
231 Quantitate, Bias Correction and Background Correction was checked for protein
232 quantification and normalization. Proteinpilot has taken into account all possible
233 modifications in the search, while adding automatic fault decoy matching function, using
234 the PSPEP (Proteomics System Performance Evaluation Pipeline Software, integrated in
235 the ProteinPilot Software) algorithm. Only proteins with at least one unique peptide and
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 236 unused value more than 1.3 (the reliability level is above 95%) were identified. These
237 identified peptides were excluded from the quantitative analysis for: (i) peptides missing
238 an iTRAQ reagent label, (ii) peptides peaks did not match the corresponding iTRAQ
239 labels, (iii) peptides with too low S/N (signal-to-noise ratio), (iv) peptides with low
240 identification confidence, (v) peptides were not unique peptide. Gene Ontology (GO,
241 http://geneontology.org/) is an internationally standardized gene function classification
242 system that provides a dynamically updated of new vocabulary to fully describe the
243 properties of genes and gene products in an organism. There was a total of three ontology
244 that describe the molecular function, cellular component, and biological process of the
245 gene in GO, respectively. When protein abundance ratio, measured in iTRAQ after
246 normalized (P-value≤0.05 for corresponding screening), the protein was considered to be
247 a significantly changed protein between different groups. The data of identified proteins
248 was then selected by the criterion（up-regulate≥1.5 or down-regulate≤0.67, P-value≤0.05）
249 for next analysis. The analysis mapped the changed proteins to the various terms from the
250 Gene Ontology database and calculates the number of proteins per term, then, functional
251 annotation for significant changed proteins of GO term. Cluster of Orthologous Groups
252 of proteins (COG, http://www.ncbi.nlm.nih.gov/COG/) is a database of orthologous
253 classification of proteins. The proteins of COG were assumed to be derived from an
254 ancestral protein, therefore, either orthologs or paralogs. Orthologs refers to proteins that
255 have evolved from vertical pedigrees from different species, and retain the same typically
256 function of the original protein. Paralogs are proteins that are derived from gene
257 duplication of a certain species and may evolve into a new and the same as the previously
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 258 relevant functions protein. To identify candidate biomarkers, we employed
259 hypergeometric test to perform GO enrichment and KEGG pathway enrichment.
260 To point out the protein-protein interactions, Search Tool for the Retrieval of Interacting
261 Genes/Proteins (STRING) was employed (http://www.string-db.org/). All other pictures
262 were drawn with R language ( http://www.r-project.org/ ).
263
264 Venn diagram
265 Venn analysis was carried out for different comparison groups of the identified
266 significant differential proteins and differential proteins involved in signaling pathways.
267 Venn diagrams were constructed online
268 ( http://bioinfogp.cnb.csic.es/tools/venny/index.html ) according to the introduction on
269 the website.
270
271 Ingenuity Pathways Analysis (IPA)
272 To obtain further insight into the functions of the identified distinct abundant
273 proteins, pathways and molecular networks between groups were analyzed using IPA
274 (Ingenuity Systems, Redwood City, CA). Briefly, identified proteins were uploaded to
275 the IPA software and mapped to their corresponding gene objects, and biological
276 functions, canonical pathways and molecular networks of gene objects were subsequently
277 evaluated and constructed based on information in the IPA database. Statistics for the
278 functional analysis were calculated automatically by the software using the right-tailed
279 Fisher’s exact test. Molecular networks generated based on ranking scores were
280 optimized to include as many proteins from input abundance profiles as possible, and to
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 281 maximize network connections. Nodes in networks were coloured, and different shapes
282 were used to represent fold changes and functional classes of genes and gene products.
283 Interactions between nodes were connected only when supported by at least one or more
284 references.
285
286 Relative quantitative PCR (qPCR)
287 The proteins (changed in abundance and had a significant impact on co-infection)
288 identified by iTRAQ were picked up and found the corresponding gene conserved
289 sequences corresponding to a gene. To validate the changes in abundance of the proteins
290 identified by iTRAQ, total mRNA was extracted from cells infected with one or both
291 viruses using TRIzol reagent (Magen) and reverse-transcribed using the SuperScript
292 First-Strand Synthesis System (Fermentas, Pittsburgh, PA) according to manufacturer
293 instructions. The GAPDH gene was used as an internal standard, and the relative
294 quantitative abundance of selected genes was assayed by real-time PCR using the SYBR
295 Premix Ex Taq (ABclonal, WuHan, China) with an ABI 7500 sequence detection system
296 (Applied Biosystems). The results were calculated as the mean value of triplicate
297 reactions. Primers used for amplification of the selected genes were shown in Table S-1.
298
299 Western Blot Assay
300 Cells were washed with PBS two times. Then treated with RIPA (EpiZyme) according to
301 the protocol (http://www.epizyme.cn/index.php?c=article&id=702). Cells were lysed in
302 ice for 30 minutes and collected the supernatant. The protein sample was quantified with
303 the BCA kit and the mass action concentration was 3.5ug/uL. The samples were mixed
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 304 with 5 plus protein loading buffer and heated in 100℃ for 5 minutes and separated on a
305 12% SDS-PAGE (10µL per lane). After electrophoresis, proteins were transfered to a
306 PVDF membrane (Millipore) at 23 volts for 0.5 hour. The PVDF membranes were
307 blocked in PBS with 5% skim milk for 1 hour. The membranes were incubated with the
308 ANXA4, LDHA, DHX9, RIC8A (ABclonal) rabbit anti-human antibody (with the
309 dilution of 1:1000) overnight at 4 °C and PCNA (rabbit anti-human) (abcam) and
310 GAPDH mouse anti-human (ABclonal) and then with the secondary antibody HRP
311 labeled goat anti-rabbit IgG (with the dilution of 1:5000, ABclonal) for 1 hour. Protein
312 levels were monitored by enhanced chemiluminescence. PNCA and GADPH were
313 employed as protein loading control.
314
315 Immunofluorescent Staining
316 The 8 treatment groups in PK15 cells were stained with the following primary antibodies:
317 rabbit anti-PCV2 (Abcam), rabbit anti-SS2-1. Treatment groups were then incubated with
318 secondary antibodies with FITC and CY3. All images were captured on a Nikon
319 fluorescent microscope equipped with a digital camera (TE2000-U, Nikon). Each
320 immunofluorescent stain was repeated three times using serial sections; negative controls
321 were included to determine the amount of background staining.
322
323 Statistical method
324 Data were presented as mean ± SD of at least three individual experiments.
325 Statistical differences between groups were analyzed by the unpaired t-test using
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 326 Microsoft Excel 2013. A value of P <0.05 was considered statistical significance.
327 Graphpad prism 7 was used to processing composite image and drawing figures.
328
329 Ethics statement
330 The procedures used for the preparation of pAb and mAb in experimental animals were
331 approved by the Institutional Animal Care and Use Committee (IACUC) of Jiangsu
332 Academy of Agricultural Sciences (Permit No. SYXK 2015-0020), in accordance with
333 the Regulations for the Administration of Affairs Concerning Experimental Animals
334 approved by the State Council of PR China.
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PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 350 RESULTS
351
352 Cell viability assay
353 PK15 cells inoculated with PCV2 (MOI=0.1) was prepared for 48h earlier, the same as
354 blank PK15 cells. PK15 cells was infected with different dose (MOI=0.1, 1, 2.5, 5 and 10)
355 and different co-culture times (2h, 4h, 6h, 12h, and 16h) of SS2 (Figure 1). The results
356 showed that the survival rate of PK15 cells was not exceed 81.9% and 78.4% within 2
357 hours, and 66.2% and 57.6% within 6 hours of SS2 infection and PCV2-SS2 co-infection
358 at the MOI=10. This figure also showed that the damage of SS2 was dose dependent and
359 time dependent. The cell basically died after 16 hours. The most distinctive feature of this
360 results indicated that PCV2 infection promoted the damage of SS2 to PK15 cells at the
361 corresponding time and MOI. Basic to the result, PK15 cells, (infected with PCV2 at the
362 MOI=1 with 48 hours and SS2-1 at the MOI=10 with 2 and 6 hours) were selected to
363 further proteomics study.
364
365 Identification and quantification of distinct abundant proteins by
366 using iTRAQ-based LC-MS/MS
367 The MS/MS spectra of unique peptides infer to the identified putative distinct abundant
368 proteins PCNA, GAPDH, ANXA4, DHX9, LDHA, RIC8A were shown in (Figure S-1).
369 A total of 4,736 proteins were identified in this project. Due to the limitations of the
370 background date annotation library, it was not all proteins in Uniprot database has
371 annotation information. There were 4,602 proteins in GO function annotation, 2457
372 proteins in COG function annotation and 2569 proteins in KEGG function annotation
18 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 373 (Figure 3 (a)). This experiment provided GO annotations, KEGG annotations and COG
374 annotations to fully reflect the function of proteins using different sources of databases,
375 revealing the biological significance of proteins in various life activities (Tables S-2 to
376 S-4, respectively).
377
378 The expression level hierarchical clustering analysis
379 Hierarchical clustering analysis brought the higher similarity proteins together intuitively.
380 Clustering the samples together by column, it could not only reflect the repeatability of
381 experimental design, but also judge the correlation between samples of different groups.
382 This analysis could provide the information that in a special condition, one or same
383 samples with high content or low content or even 0 of one certain or some proteins. The
384 results showed all the 1184 significant difference proteins of compilations of the 8
385 different groups (samples) (Figure 3 (d)). This horizontal of hierarchical clustering
386 analysis showed that, according to the degree of protein proximity, the order of different
387 groups was Blank-2h, Blank-6h, PCV2-2h, PCV2-6h, SS2-2h, PCV2-SS2-2h, SS2 and
388 PCV2-SS2-6h. Simultaneously, it reflected the similarity degree of proteins between
389 different treatment groups. Among them, 2 blank groups were closed, 2 PCV2 single
390 infected groups (with the treatment time 50 hours and 54 hours) were closed and the SS2,
391 PCV2-SS2 treatment groups (with the treatment time 2 hours and 6 hours) were closed.
392 The vertical 1184 proteins were hierarchical clustered according to the similarity
393 correlations and the proteins ID (UniProt) were listed in Figure S-2. Proteins in the same
394 cluster showed similar abundance, similar characteristics, or participate in the same
395 cellular process.
19 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 396
397 GO annotation
398 We performed a GO functional annotation analysis on all identified proteins, and for the
399 GO class involved in the three ontologies (cellular component, biological process,
400 molecular function). At the same time, make a chart (Figure 3 (b)) and omit the GO class
401 without the corresponding protein. For different groups/samples Blank-2h, Blank-6h,
402 PCV2-2h, PCV2-6h, SS2-2h, SS2-6h, PCV2-SS2-2h and PCV2-SS2-6h, GO annotation
403 were performed GO annotation analysis to compare the treatment groups on differential
404 proteins like PCV2-2h: Blank-2h, PCV2-SS2-2h: Blank-2h, SS2-2h: Blank-2h, PCV2-6h:
405 Blank-6h, PCV2-SS2-6h: Blank-6h and SS2-6h: Blank-6h (Figure 4). As showed in
406 Figure 4, as stated in GO classification, all identified proteins in different groups were
407 generally divided into three classes (biological process, cellular component, and
408 molecular function) and further subcategorized into 45 hierarchically structured GO
409 classifications for detailed investigation. It showed the proportion of up-regulated and
410 down-regulated proteins in each subcategory. The results showed that biological process
411 contained cellular process, metabolic process, biological regulation, regulation of
412 biological process, cellular component organization or biogenesis, response to stimulus,
413 multicellular organism process, development process, localization, negative regulation of
414 biological process, establishment of localization, positive regulation of biological process,
415 signaling, immune system process, death, locomotion, reproduction, reproductive process,
416 cell proliferation , biological adhesion, growth, viral reproduction, multi-organism
417 process, cell killing, rhythmic process, while cellular contained cell, cell part, organelle,
418 macromolecular complex, membrane enclosed lumen, extracellular region, extracellular
20 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 419 region part, synapse, synapse part and molecular function contained binding, catalytic
420 activity, enzyme regulator activity, transporter activity, structure molecular activity,
421 molecular transducer activity, antioxidant activity, channel regulator activity, electron
422 carrier activity, translation regulator activity. For different groups, the distribution of
423 major components was similarity, but not the same.
424
425 COG annotation
426 We compared the identified proteins with the COG database, predict the possible
427 functions of these proteins and perform functional classification statistics on them. Figure
428 3 (c) indicated all these proteins showed 24 functions, and the numbers is Tables S-4. The
429 most abundant proteins were posttranslational modification, protein turnover, chaperones
430 proteins and the least abundant proteins were nuclear structure proteins. This result
431 provoked us to focus on the organelle and organelle part relevant differential expressed
432 proteins.
433
434 GO pathway analysis
435 All the identified proteins had been performed pathway analysis according to the KEGG
436 database and found the most import ant biochemical metabolic pathways and signal
437 transduction pathways involved in protein. Figure 5 shows the metabolic pathways and
438 signal transduction pathways of proteins between different groups. It also illustrated the
439 ratio of up-regulated and down-regulated proteins to different metabolic and signal
440 transduction pathways. This result showed that metabolic pathways is the mainly
441 pathways among these groups. In PCV2 groups, the mainly up-regulated and
21 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 442 down-regulated pathways existed big difference. The up-regulated proteins involved in
443 metabolic pathways, glycolysis/gluconeogenesis, microbial metabolism in diverse
444 environments, aminoacyl-tRNA biosynthesis, oocyte meiosis, pentose phosphate pathway,
445 proteasome, Wnt signaling pathway, long-term depression, aldosterone regulated sodium
446 reabsorption, ribosome, purine metabolism, pyruvate metabolism, cysteine and
447 methionine metabolism, and pyrimidine metabolism, while the down-regulated proteins
448 involved in metabolic pathways, huntington’s disease, lysine degradation, insulin
449 signaling pathway, phagosome, microbial metabolism in diverse environments,
450 endocytosis, oxidative phosphorylation, spliceosome, gastric acid secretion, protein
451 processing in endoplasmic reticulum, focal adhesion, ECM-receptor interaction, pathway
452 in cancer, Alzheimer’s disease, toxoplasmosis, small cell lung cancer and cell adhesion
453 molecules. In PCV2-SS2 groups, the up-regulated proteins involved in metabolic
454 pathways, ribosome, regulation of actin cytoskeleton, tight junction, microbial
455 metabolism in diverse environments, protein processing in endoplasmic reticulum,
456 spliceosome, alanine, aspartate and glutamate metabolism peroxisome, pathogenic
457 Escherichia coli infection, aminoacyl-tRNA biosynthesis, pathways in cancer,
458 toxoplasmosis, protein processing in endoplasmic reticulum, complement and
459 coagulation cascades, focal adhesion and amoebiasis, while the down-regulated proteins
460 involved in metabolic pathways, microbial metabolism in diverse environments, focal
461 adhesion, regulation of actin cytoskeleton, ECM-receptor interaction, RNA transport,
462 spliceosome, dilated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy
463 (ARVC), hypertrophic cardiomyopathy, glycolysis/gluconeogenesis, regulation of actin
464 cytoskeleton, purine metabolism, pyruvate metabolism, lysosome, pentose phosphate
22 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 465 pathway, cysteine and methionine metabolism. In SS2 groups, the up-regulated proteins
466 involved in spliceosome, protein processing in endoplasmic reticulum, metabolic
467 pathways, staphylococcus aureus infection, ubiquitin mediated proteolysis, complement
468 and coagulation cascades, aminoacyl-tRNA biosynthesis, malaria, tight junction and
469 regulation of actin cytoskeleton and pathway in cancer, ECM-recepror interaction, focal
470 adhesion, cell cycle, DNA replication, small cell lung cancer, ramoebiasis, viral
471 myocarditis, while the down-regulated proteins involved in metabolic pathways microbial
472 metabolism in diverse environments, glycolysis/gluconeogenesis, pathogenic Escherichia
473 coli infection, protein processing in endoplasmic reticulum, regulation of actin
474 cytoskeleton, glutathione metabolism, pathways in cancer, endocytosis, tight junction,
475 proteasome, purine metabolism, glutathione metabolism, pyruvate metabolism, cysteine
476 and methionine metabolism. This result showed that there were the same and the different
477 pathways among these main signaling pathways of different groups. The detail
478 information of the differential expression proteins in the major signaling pathways were
479 listed in the attached table S-5. These protein changes objectively and positively reflected
480 the material basis of PCV2 exacerbating the damage of Streptococcus suis to PK15 cells.
481 But how this effect was linked needs further analysis and verification
482
483 Venn diagram analysis
484 In order to further find the connection between these groups, Venn diagram analysis was
485 performed. In other words, identified and pathways involved with significant
486 differences in p2/b2, s2/b2, ps2/b2, p6/b6, s6/b6, and ps6/b6 proteins were analyzed
487 using a Venn diagram (Figure 6). There were 5 common proteins in the treatment of 2
23 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 488 hours groups (p2/b2, s2/b2 and ps2/b2), and they were 40S ribosomal protein S9 (RPS9),
489 Serum albumin (ALB), RIC8 guanine nucleotide exchange factor A (RIC8A), Argonaute
490 3 (AGO3) and Oxysterol-binding protein (OSBPL1A), which participate in
491 Aminoacyl-tRNA biosynthesis, Ribosome pathway. This result showed that the PCV2
492 and SS2 could interfere protein synthesis together in the SS2 early infection stage. And
493 there were 15 common proteins in the treatment of 6 hours groups (p6/b6, s6/b6, and
494 ps6/b6), and they were Laminin subunit alpha 5 (LAMA5), Uncharacterized protein
495 LAMC1, Laminin subunit beta 1 (LAMB1), L-lactate dehydrogenase (LDHA), Splicing
496 factor proline and glutamine rich (SPFQ), Annexin (ANXA4), Cathepsin D protein
497 Fragment, Carbonyl reductase 3 (CBR3), 60S ribosomal protein L6 (RPL6), Integrin beta
498 (ITGB1), Serum albumin (ALB), Aminoacylase-1 (ACY1), Cathepsin D protein, Gam
499 ma-tubulin complex component (TUBGCP2), Replication initiator 1 (REPIN1), Golgin
500 A3 (GOLGA3) and Alpha-1,3-mannosyl-glycoprotein
501 4-beta-N-acetylglucosaminyltransferase B (MGAT4B). This result showed that the 2
502 pathogens induced more serious cell damage proteins including in Toxoplasmosis,
503 Amoebiasis, Shigellosis, Leishmaniasis, Prion diseases, Pathogenic Escherichia coli
504 infection and Small cell lung cancer pathways after 6 hours infection. Simultaneously, a
505 total of 199 pathways were categorized (Table S-6), among then, 44 same pathways were
506 involved in all 6 groups, 55 same pathways in 5 groups, 36 same pathways in 4 groups,
507 26 same pathways in 3 groups and 20 same pathways in 2 groups and 20 pathways just in
508 single groups. This showed that the distribution of proteins was very widely in both
509 single infection groups and co-infection groups. And the data indicated that the number
510 of down-regulated proteins was significantly higher than the number of up-regulated
24 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 511 proteins in these six treatment groups (Figure 6). This data also could see that the PK15
512 cells infected with PCV2 before were not as violent reaction as they single infected with
513 SS2, but PCV2 pre-infected cells had lower survival numbers when SS2 invaded.
514
515 Ingenuity Pathways Analysis (IPA)
516 In response to the above results, we selected the proteins, related to PCV2 infection and
517 SS2 infection early and intermediate stages, and the preformed STRING analysis to
518 explore protein interaction network (Figure 7) and ingenuity pathways analysis. But some
519 proteins without information in database were not figured. In figure 10, We draw a
520 network diagram that interacts with 40S ribosomal protein S9 (RPS9), RIC8 guanine
521 nucleotide exchange factor A (RIC8A), 60S ribosomal protein L6 (RPL6),
522 Alpha-1,3-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase B (MGAT4B)
523 and Gamma-tubulin complex component (TUBGCP2). This figure expressed that the
524 interaction relationship in inner cells and force magnitude of interaction of the differential
525 proteins. The proteins, participated in the regulation of PK15 infected by the two
526 pathogens, were draw in corresponding signaling pathway with their up-regulated and
527 down regulated information at the link of
528 (file:///Users/tanjimin/Desktop/pcv2-ss2-pk-ITRAQ%20analysis%20Project_data/2.Enric
529 hment/Pathway_enrichment/PCV2_2h-Blank_2h_Pathway_enrichment/PCV2_2h-Blank
530 _2h.htm) and
531 (file:///Users/tanjimin/Desktop/pcv2-ss2-pk-ITRAQ%20analysis%20Project_data/2.Enric
532 hment/Pathway_enrichment/PCV2_6h-Blank_6h_Pathway_enrichment/PCV2_6h-Blank
533 _6h.htm#gene21).
25 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 534 This result indicated that PCV2 and SS2 co-infection and the two pathogens single
535 infection to PK15 cells, respectively, there were some key function/pathways participate
536 in. And the common pathways were ribosome and aminoacyl-tRNA biosynthesis.
537 Simultaneously, they also via ECM-receptor interaction pathway interacted with the cell
538 membrane, so as to achieved the purpose of pathogenic. The pathway maps could help to
539 further explore the pathogenic mechanism of the two pathogens.
540
541 Relative quantitative PCR (qPCR)
542 Based on proteomics data and the comparative analysis. The differential proteins were
543 selected and according to those protein sequences and gene sequences, we designed the
544 relative fluorescence quantitative primers of these genes (Table S-7). PCR results showed
545 that the expression of mRNA was not all the same with the result of proteomics data. The
546 proteins with large variation folds and the reference genes showed consistency with
547 mRNA results trend among different groups. And the iTRAQ data showed distinct
548 differencial proteins were consistency with the qPCR data. This conclusion could be
549 reflected in the results of LDHA, ANXA4, RIC8A, DHX9 mRNA experiments (Figure
550 8).
551
552 Western Blot Assay
553 In one aspect, six proteins were detected by western blot to verify the proteomic and
554 bioinformational data (Figure 9). And the results instrumented that the western blot
555 results of LDHA, ANXA4, RIC8A, DHX9 was consistency with the proteomic data and
26 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 556 bioinformational data. Simultaneously, this result pointed out several co-infection key
557 proteins of PCV2 infection, SS2 infection, and PCV2-SS2 co-infection key proteins.
558 3.11 Fluorescence experiment of the two pathogens PCV2 and SS2 in PK15 cells
559 As showed in the Figure 10, PCV2 and SS2 were pictured in fluorescence microscope.
560 Meanwhile, gram stain was used for SS2 for comparison with immunofluorescence. This
561 data indicated that PK15 cells were more adherent to bacteria after infected with PCV2
562 for the amount of SS2 in PCV2 infected cells in larger than PCV2 without cells.
563 Fluorescence experiment verified the two pathogens were executed in all this experiment.
564
565
566
567
568
569
570
571
572
573
574
575
27 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 576 DISCUSSION
577 When pathogens infect organisms, tissues, and cells, host changes were a complex
578 process, or rather, it was a collection concept. PCV2 could persist existing in this large
579 heterogenous family of myeloid cells (like monocyte, macrophage and dendritic cell)
580 without replicating or being degraded which like a “Trojan horse” 43 and PK15 cell was
581 used to culture and isolation of viruses of PCV. PCV2 could infect many cells in vivo,
582 but it caused the slightest lesions in porcine kidney cells, perhaps, it was the response to
583 the infection rather than the infection itself that results in the most damage, death and a
584 general decline into catabolic state to the bystander cells 2,10,44-46. PCV2 existing research
585 showed that PCV2 induce apoptosis and autophagy by HSP70, HPS90, ROS, MKRN1,
586 pPirh2 47. In the early infection stage virus could induce pro-inflammatory cytokine
587 expression 48. PCV2 is a negative modulates virus in vitro 49. Type I and type II
588 interferons induce cellular protein alteration and increases replication of porcine
589 circovirus type 2 in PK15 cells 50. PCV2 induces IFN beta expression through increased
590 expression of genes involved in RIG-I and IRF7 signaling pathways 51. Study also
591 showed PCV2 lead to thymocyte selection dysregulation 43.
592 Extra interleukin-1 supplementation could help newborn piglets resist the infection of
593 streptococcus suis by the may augment nonspecific immune function 52. Studied literature
594 had reported that streptococcus suis has albumin binding activity and plasminogen
595 binding activity and the additional of albumin and plasminogen could increase the
596 virulence of Streptococcus suis to mouse 53,54. And some reported that extracellular
597 proteins like cellular fibronectin and collagen I, III and V, fibrin, vitronectin and laminin
598 could bind to Streptococcus suis 55. Heat-killed capsular of Streptococcus suis type 2
28 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 599 strains could stimulate tumor necrosis factor alpha and interleukin-6 production by
600 murine macrophages 56. It was said that the related diseases caused by Streptococcus suis
601 start from the colonization of bacteria to nasal mucosal epithelial cells, and then follow
602 with either the flow of respiratory tract or the bloodstream 57. The mechanisms of bacteria
603 how to be involved in the access that from the bloodstream to the central nervous system
604 were unknown. Simultaneously, it was possible that epithelial cells of the choroid plexus
605 also play an important role in the pathogenesis of the meningitis. The common epithelial
606 cell lines in the laboratory include LLC-PK1, PK (15), A549, HeLa and MDCK, etc. It
607 was reported that SS2 was able to adhere to but not to invade epithelial cells, and the
608 haemolysin produced by S. suis serotype 2 was responsible for a toxic effect observed on
609 epithelial cells, that is to say, it was possible that suilysin positive S. suis strains use
610 adherence and cell injury, as opposed to direct cellular invasion to epithelial cells 57. Cell
611 death, caspase activation, and high mobility group box 1 (HMGB1) protein release of
612 porcine choroid plexus epithelial cells during streptococcus suis infection in vitro 58.
613 As said above, both pathogens caused significant harm to pigs or even human at clinical.
614 The pity was that predecessor studies were confined to understand the infection and
615 co-infection of the two pathogens from the main thought of a single molecule. We
616 explored the proteomics changes of PK15 cells when co-infected with the two pathogens.
617 As the case of a complex co-infection, experiment was starting from a single cell and
618 using proteomics and bioinformatics to find the differential proteins of different groups,
619 which directly and objectively presented the complex protein changes, suggested possible
620 mechanisms for further study.
29 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 621 Our results demonstrated that PCV2 cause apoptosis for it induce the chaperonin
622 containing tailless complex polypeptide 1 decrease 59 and supplemented pathways like
623 Wnt/Ca2+ , and SS2 could binding to cell extracellular secreted protein 60,61 though
624 EMC-receptor interaction. PCV2 caused cell catabolism was greater than anabolic, while
625 SS2 caused a violent cell response. For PCV2-SS2 infection group, in the early stage of
626 SS2 infection, for the before infection of PCV2, the response of cells was compensatory,
627 but in the middle infection of SS2, the toxic protein (of which the effect was similar to
628 amoeba, toxoplasma and carcinogen) was further damage to cells and lead to the decline
629 cells activity. SS2 single infection also caused cell damage, but cell could do some
630 defense not only in a compensatory or passive state.
631 Simultaneously, the data indicated that PCV2 not only lead to diseases relating to
632 metabolism, but also lead to neurological disease, which may the main reason that PCV2
633 could cause PWMS and PDNS 62-64. In the case that PCV2 made the cells in a passive
634 state, it was more convenient for us to observe the main mechanism of action of
635 Streptococcus suis in PK15 cells, we found that SS2 could decrease the proteins of tight
636 junction, actin cytoskeleton and endoplasmic reticulum which made the cell more
637 dispersive and this derivation could be seen in the figure 13 (The more cells were
638 dispersed, the more SS2 was adsorbed, and promoted each other), which may an evidence
639 that SS2 could pass the blood-brain barrier 65,66. And the SS2 migration mechanism
640 in cells was worth to be further study.
641 Cell survival results indicated that PCV2 increased the cell mortality when SS2 infected
642 (Figure 1). But the proteomics showed that PCV2 decreased the cell response degree
643 when SS2 infected (Figure 9). This may be explained by the infection of PCV2 that left
30 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 644 the cells in a state of slaughter by SS2 without defense. This result also inspired curiosity
645 if these two pathogens were infected on immune cells as SS2 could cause a rapid and
646 intense immune response including inflammation response in the early stage. But after all,
647 they are all pathogens.
648 We listed the proteins that changed in different groups of PK15 cells and made a
649 comparative analysis and fund some common grounds, the two pathogens existed some
650 virulence factor that damage the cells like some pathogenesis of parasites. But the detail
651 proteins of PCV2 and SS2 are don’t know base to our experiment. These common
652 proteins including cell membrane receptor proteins, ribosome-associated proteins,
653 Transport RNA-associated proteins, Lysosome, Purine metabolic, N-Glycan biosynthesis
654 proteins. And we performed an intracellular network interaction analysis about then. In
655 order to facilitate subsequent research, protein pathway analysis was performed on all
656 differential proteins. For reliability, qPCR experiments and Western blot experiments
657 validate our data and immunofluorescence experiments showed our pathogens.
658 Our experiments revealed that 1 point: proteomics was a good science technology to
659 understand pathogenic infections and pathogenic mechanisms, 2 point: the main reason of
660 PCV2 infection exacerbated the cell survival caused by SS2 infection for PCV2 maked
661 cells in a disintegrated state and the proteins in the cell was reduced and the cell
662 metabolic ability was weakened, so it was in a naive state and lacks defense capabilities,
663 3 point: except nutritional competition, SS2 can stimulate cells very strongly and produce
664 very strong changes in proteins, which was too harmful to cell.
665 The changed proteins or mainly pathogenic mechanism of PCV2 were mainly including
666 metabolic pathways, hunting’s disease, lysine degradation, insulin signaling pathway,
31 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 667 phagosome, microbial metabolism in diverse environments, endocytosis, oxidative
668 phosphorylation, spliceosome and gastric acid secretion. While, SS2 mainly pathogenic
669 mechanism or changed proteins were including spliceosome, protein processing in
670 endoplasmic reticulum, metabolic pathways, staphylococcus aureus infection, ubiquitin
671 mediated proteolysis, complement and coagulation cascades, aminoacyl-tRNA
672 biosynthesis, malaria, tight junction and regulation of actin cytoskeleton. When PK15
673 cells were co-infected with the two pathogens and SS2 was the main reason made the
674 cells to death in a short time.
675 Animals were always exposed to microorganisms or pathogenic environments. This study
676 used PK15 cells as a model to explored a series of protein changes produced by PCV2
677 and SS2 single and co-infection. Definitely, when pathogens encountered to their host, a
678 complex set of intracellular and extracellular interactions determines the outcome of the
679 infection. Understanding the interactions between those host animal or cell and pathogens
680 was critical to developing treatments and preventive measures against infectious diseases.
681 So, the next study about the special proteins of pathogens-host interactions worth to be
682 further explore.
683
684 CONCLUSION
685 Conflict of interest
686 None to declare.
687
688 ACKNOWLEDGEMENTS
32 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
PCV2 and SS2 co-infection proteomic study of Porcine Kidney cell 15 689 This work was supported by the National Natural Sciences Foundation of
690 China (31972679), and Research Project from Jiangsu Key Laboratory for Food Quality
691 and Safety-State Key Laboratory Cultivation Base, Ministry of Science and
692 Technology(2019SY001). The authors would like to express their appreciation to Wuhan
693 GeneCreate Biological Engineering Co., Ltd. for their contributions to the bioinformatics
694 support for this project.
695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728
33 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
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42 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure 1 | The cell viability assay when SS2 single infection and with PCV2 co-
infection. The ordinate indicated the cell survival rate/viability, and the abscissa was
the time that SS2 interaction with PK15. The infection MOI of SS2 was divided into
six groups, they were 0, 0.1, 1, 2.5, 5, and 10, respectively, represented by dark to light
blue. bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure 2 | Proteomics implementation schemes for different groups using iTRAQ.
There were A, B, C, D, E, F, G and H 8 groups were ready to be used for iTRAQ and
proteomics analysis. The same amounts of samples were extracted from these 8 groups
to form an internal standard group named I. These 9 groups conducted two ITRAQ
experiments named ITRAQ E1 and E2 which all including I group. And then the
experiments were analyzed in accordance with the procedure. bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure 3 | Number of total proteins in GO, COG and KEGG database,
distribution of total proteins in GO and COG and cluster analysis of total
proteins for 8 groups. (a), the total proteins identified in GO, COG, and KEGG
database. (b), GO analysis of 4602 proteins including biological process, cellular bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. process and molecular function which also including different secondary functional
classifications and proportions marked in different colors. (c), COG function
classification of 2457 proteins and different colors represents different COG functions
and proportions. (d), the cluster analysis of identified proteins in 8 groups, of which
the ordinate indicated the near and far relationships according unique peptide of
different identified proteins and the abscissa indicated proximity of different groups.
Green color represented proteins down-regulated, red color represented proteins up-
regulated and the color shade degree indicated the degree of up and down regulated.
Figure 4 | GO analysis of differential proteins in treatment groups to blank bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. groups. The ordinate indicated the percentage of protein, the functional classifications
in biological process, cellular component and molecular function, respectively. Green
color and yellow color represented down and up regulated, respectively. The groups
included PCV2-2h:Blank-2h, PCV2-6hBlank-6h, PCV2-SS2-2h:Blank-2h, PCV2-
SS2-6h:Blank-6h, SS2-2h:Blank-2h and SS2-6h:Blank-6h.
bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure 5 | KEGG analysis of differential proteins in treatment groups to blank
groups. There were 6 differential proteins groups including PCV2-2h:Blank-2h,
PCV2-6hBlank-6h, PCV2-SS2-2h:Blank-2h, PCV2-SS2-6h:Blank-6h, SS2-2h:Blank-
2h and SS2-6h:Blank-6h. There were up-regulated and down-regulated differential
proteins in the 6 groups. Each pie chart contained the percentage of the top ten major
pathways (listed in order from high to low below).
bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure 6 | The Venn analysis of differential proteins in treatment groups to blank
groups of KEGG data. According to 2 time points of 2h and 6h, the 6 groups of
differential proteins were divided into two groups and the up-regulated and down-
regulated differential proteins were performed Venn analysis, respectively. The
abscissa indicated 6 comparison groups and the ordinate represented the number of
proteins involved in pathways and the number of proteins involved in pathways maps,
respectively. bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure 7 | The proteins interaction analysis by STING and the proteins were
produced by PCV2 and SS2 co-infection and stimulating PK-15 cells. There were
5 proteins among 14 co-infection proteins existing proteins interaction maps and they
were RPS9, TUBGCP2, RPL6, MGAT4B and RIC8A. The force between proteins
was represented by different line segments. Network nodes represented proteins. Red
nodes represented query proteins and first shell of interactors and white nodes
represented second shell of interactors. Empty nodes represented proteins of unknown
3D structure and filled nodes represented some 3D structure was known or predicted.
And the edges represented protein-protein associations, the fluorescent green
represented activation, the blue represented binding, the light green bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. represented phenotype, the dark represented reaction, the red represented
inhibition, the purple represented catalysis, the light red represented
posttranslational modification and the yellow represented transcriptional
regulation. The represented positive, the represented negative and the
represented unspecified.
Figure 8 | The qPCR verification and the proteins changed folds of iTRAQ data. bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The left of the figure was qPCR data and the right of the figure was iTRAQ data. The
* and **represented p<0.05 and p<0.01. Dark to light gray represented groups Blank,
PCV2, PCV2-SS2 and SS2.
Figure 9 | Western blot verification assay. The left of this figure was western blot
data of PCNA, GAPDH, ANXA4, DHX9, LDHA and RIC8A, as well as the right of
this figure was the statistical data of western blot. The ordinate of the histogram was
the gray value of proteins and the abscissa of the histogram were 8 groups. bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure 10 | The pictures of SS2 and PCV2 fluorescence stain in fluorescence
microscope and Gram stain in microscope. The fluorescence stain of SS2 was
marked by CY3 and PCV2 was marked by FITC. Meanwhile, SS2 was also done with
Gram stain. a, the blank cells without SS2 infection. b, SS2 infected cells with 2 hours
and was showed red fluorescence in fluorescence microscope. c, PCV2 and SS2 co-
infected cells with 2 hours and was showed red fluorescence in fluorescence
microscope. d, SS2 infected cells with 6 hours and was showed red fluorescence in
fluorescence microscope. c, PCV2 and SS2 co-infected cells with 6 hours and was bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. showed red fluorescence in fluorescence microscope. f, SS2 was stained by Gram
stain and showed in microscope. g, the blank cells without PCV2 infection. h, PCV2
infected cells and was showed green fluorescence in fluorescence microscope. c,
PCV2 and SS2 co-infected cells and was showed green fluorescence in fluorescence
microscope. The length of the ruler in the pictures were 20 µm. bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure S-1 | The mass spectrum of protein unique peptide and the mass-to-charge ratio of amino acid in the unique peptide. The mass spectrum
figures showed the unique peptide corresponding to a protein, and the tables showed the mass-to-charge ratio of each amino acid in the unique peptide.
This figure showed 6 unique peptides of 6 proteins (PCNA, GAPDH, ANXA4, DHX9, LDHA and RIC8A). -3.0 1:1 3.0 Blank-2h Blank-6h PCV2-2h PCV2-6h SS2-2h PCV2-SS2-2h SS2-6h PCV2-SS2-6h
tr|F2Z5B4|F2Z5B4_PIG tr|F1RYA3|F1RYA3_PIG tr|I3LS60|I3LS60_PIG tr|A0A287ASA9|A0A287ASA9_PIG tr|A0A287BMV2|A0A287BMV2_PIG tr|A0A286ZLF6|A0A286ZLF6_PIG tr|I3LJD8|I3LJD8_PIG tr|K7GQ86|K7GQ86_PIG tr|F1RKU4|F1RKU4_PIG tr|F1RW08|F1RW08_PIG tr|F1S3C7|F1S3C7_PIG tr|F1S8K5|F1S8K5_PIG tr|A0A286ZRL1|A0A286ZRL1_PIG tr|F1SP83|F1SP83_PIG tr|F1S210|F1S210_PIG tr|F1RZU4|F1RZU4_PIG tr|K7GPD4|K7GPD4_PIG tr|A0A287AZJ9|A0A287AZJ9_PIG tr|A0A287ATG0|A0A287ATG0_PIG tr|F1SCV1|F1SCV1_PIG tr|F1S197|F1S197_PIG tr|F1RFG1|F1RFG1_PIG tr|A0A287AK91|A0A287AK91_PIG tr|K9IVV3|K9IVV3_PIG tr|A0A287A309|A0A287A309_PIG tr|A0A287BRH5|A0A287BRH5_PIG tr|F1RQZ9|F1RQZ9_PIG tr|I3LHC4|I3LHC4_PIG tr|F1RU39|F1RU39_PIG tr|A0A288CFU9|A0A288CFU9_PIG tr|A0A287B3P6|A0A287B3P6_PIG tr|F2Z546|F2Z546_PIG tr|A0A286ZX08|A0A286ZX08_PIG tr|A0A287BQW3|A0A287BQW3_PIG tr|A0A286ZW72|A0A286ZW72_PIG tr|A0A286ZZB7|A0A286ZZB7_PIG tr|A7TX82|A7TX82_PIG tr|A0A287A527|A0A287A527_PIG tr|A0A287A9I8|A0A287A9I8_PIG sp|Q2YGT9|RL6_PIG tr|I3LSD3|I3LSD3_PIG tr|A0A287A6Z8|A0A287A6Z8_PIG tr|F1RFI1|F1RFI1_PIG tr|A0A0B8RT23|A0A0B8RT23_PIG sp|Q29361|RL35_PIG tr|A0A286ZQA5|A0A286ZQA5_PIG tr|F1SJJ5|F1SJJ5_PIG tr|A0A286ZJQ4|A0A286ZJQ4_PIG tr|F1RT46|F1RT46_PIG tr|I3LKA3|I3LKA3_PIG tr|I3LT81|I3LT81_PIG tr|A0A287B4P8|A0A287B4P8_PIG tr|F1SIX3|F1SIX3_PIG tr|A0A286ZIC1|A0A286ZIC1_PIG tr|K7GQ95|K7GQ95_PIG tr|A0A287B5V1|A0A287B5V1_PIG tr|A0A287ASD0|A0A287ASD0_PIG tr|F1S9K5|F1S9K5_PIG tr|I3LD43|I3LD43_PIG tr|A0A286ZL51|A0A286ZL51_PIG tr|F2Z5C7|F2Z5C7_PIG tr|I3LR32|I3LR32_PIG tr|F1SSA6|F1SSA6_PIG tr|I3LK68|I3LK68_PIG tr|F1SK00|F1SK00_PIG tr|A0A287BBQ0|A0A287BBQ0_PIG tr|F1S297|F1S297_PIG tr|F1SEN2|F1SEN2_PIG sp|M3TYT0|ROCK2_PIG tr|F1SNW8|F1SNW8_PIG sp|Q767L8|MDC1_PIG tr|F1S069|F1S069_PIG tr|F1SSS0|F1SSS0_PIG tr|F1RTZ0|F1RTZ0_PIG tr|I3L7B7|I3L7B7_PIG tr|F1RS45|F1RS45_PIG tr|A0A287B3T2|A0A287B3T2_PIG tr|A0A287A9Q7|A0A287A9Q7_PIG tr|A0A287AG72|A0A287AG72_PIG tr|A0A287BJR3|A0A287BJR3_PIG tr|A0A286ZMH0|A0A286ZMH0_PIG tr|A0A286ZN79|A0A286ZN79_PIG tr|A0A287A784|A0A287A784_PIG tr|A0A287BAI8|A0A287BAI8_PIG tr|F1SGG1|F1SGG1_PIG tr|F1S0J8|F1S0J8_PIG tr|A0A287A2J4|A0A287A2J4_PIG tr|A0A286ZPH3|A0A286ZPH3_PIG tr|F1SCW5|F1SCW5_PIG tr|C5I4T6|C5I4T6_PIG tr|A0A287BTG4|A0A287BTG4_PIG tr|A0A287A080|A0A287A080_PIG tr|A0A287AQH3|A0A287AQH3_PIG tr|F1SG41|F1SG41_PIG sp|Q8MJ76|FETA_PIG tr|F1RUN2|F1RUN2_PIG tr|F6PS90|F6PS90_PIG sp|P09571|TRFE_PIG tr|F1SQT3|F1SQT3_PIG tr|I3LSG1|I3LSG1_PIG tr|A0A287AAF5|A0A287AAF5_PIG tr|I3LDS3|I3LDS3_PIG tr|A0A287BAZ8|A0A287BAZ8_PIG tr|F1S029|F1S029_PIG tr|I3LPL7|I3LPL7_PIG sp|P11607|AT2A2_PIG tr|F1S596|F1S596_PIG tr|F1S441|F1S441_PIG tr|A0A287BQI7|A0A287BQI7_PIG tr|A0A287ADJ2|A0A287ADJ2_PIG tr|A0A287B5Q7|A0A287B5Q7_PIG tr|A0A287AVR0|A0A287AVR0_PIG tr|F1SMZ7|F1SMZ7_PIG tr|Q30E20|Q30E20_PIG tr|F1RZQ3|F1RZQ3_PIG tr|I3LD13|I3LD13_PIG tr|I3LU08|I3LU08_PIG tr|F1S2F6|F1S2F6_PIG tr|A0A287ABB8|A0A287ABB8_PIG tr|F1S8S6|F1S8S6_PIG tr|F1SJF9|F1SJF9_PIG tr|F1S227|F1S227_PIG tr|A0A287A7G8|A0A287A7G8_PIG tr|F1SCF5|F1SCF5_PIG tr|A0A286ZZ97|A0A286ZZ97_PIG tr|F1ST49|F1ST49_PIG sp|P00348|HCDH_PIG tr|I3LUR5|I3LUR5_PIG tr|A0A287BSI6|A0A287BSI6_PIG tr|A0A286ZPT0|A0A286ZPT0_PIG tr|A0A286ZT52|A0A286ZT52_PIG tr|I3LP78|I3LP78_PIG tr|I3LUM9|I3LUM9_PIG tr|A0A287AVQ1|A0A287AVQ1_PIG tr|F1SB53|F1SB53_PIG tr|F1RJX8|F1RJX8_PIG tr|A0A286ZU22|A0A286ZU22_PIG tr|A0A286ZQU7|A0A286ZQU7_PIG sp|Q19AZ8|THRB_PIG tr|I3LTB8|I3LTB8_PIG tr|I3LN42|I3LN42_PIG tr|E3W6T5|E3W6T5_PIG tr|F1S0D9|F1S0D9_PIG tr|F8UV46|F8UV46_PIG tr|I3LD72|I3LD72_PIG tr|A0A287B893|A0A287B893_PIG tr|K9J6K6|K9J6K6_PIG tr|I3LHZ9|I3LHZ9_PIG tr|F1RMF9|F1RMF9_PIG tr|A0A286ZR21|A0A286ZR21_PIG tr|A0A287BMY7|A0A287BMY7_PIG tr|I3LGD9|I3LGD9_PIG tr|A0A287AEH1|A0A287AEH1_PIG tr|A0A287BIG0|A0A287BIG0_PIG tr|F1SAE9|F1SAE9_PIG tr|F1S8Y8|F1S8Y8_PIG tr|K7GM40|K7GM40_PIG sp|P02067|HBB_PIG tr|F1SM61|F1SM61_PIG tr|I3LJL2|I3LJL2_PIG tr|F1S4Q8|F1S4Q8_PIG tr|Q2XQ99|Q2XQ99_PIG tr|F1RGX4|F1RGX4_PIG tr|A5Z221|A5Z221_PIG tr|F1RIG4|F1RIG4_PIG tr|I3LJ79|I3LJ79_PIG tr|A0A0B8S002|A0A0B8S002_PIG tr|F1RKZ6|F1RKZ6_PIG tr|F1RJW8|F1RJW8_PIG tr|I3LPB5|I3LPB5_PIG tr|A5A788|A5A788_PIG tr|F1RJR9|F1RJR9_PIG tr|F1SI06|F1SI06_PIG tr|F1RX17|F1RX17_PIG tr|A0A287AU52|A0A287AU52_PIG tr|I3LS62|I3LS62_PIG tr|I6L6E1|I6L6E1_PIG sp|Q95250|PGRC1_PIG tr|K9IVK2|K9IVK2_PIG tr|A0A287AMA9|A0A287AMA9_PIG tr|I6L631|I6L631_PIG tr|A0A287A901|A0A287A901_PIG tr|A0A287BPF1|A0A287BPF1_PIG tr|A0A286ZP73|A0A286ZP73_PIG tr|F1S0H7|F1S0H7_PIG tr|K7GR19|K7GR19_PIG tr|F1S4U6|F1S4U6_PIG tr|I3LV99|I3LV99_PIG tr|K9J6M4|K9J6M4_PIG tr|K9J6K2|K9J6K2_PIG tr|A0A287BMJ2|A0A287BMJ2_PIG tr|F1RHH8|F1RHH8_PIG tr|A0A287A0F4|A0A287A0F4_PIG tr|A0A287B600|A0A287B600_PIG tr|I3L7T4|I3L7T4_PIG tr|F1SJG2|F1SJG2_PIG tr|A0A2C9F3A3|A0A2C9F3A3_PIG tr|F1SUD6|F1SUD6_PIG tr|I3LFK8|I3LFK8_PIG tr|A0A287ACN4|A0A287ACN4_PIG tr|Q19QT0|Q19QT0_PIG tr|F1RUP7|F1RUP7_PIG tr|A0A287BF68|A0A287BF68_PIG sp|P00506|AATM_PIG tr|A5GFU7|A5GFU7_PIG tr|I3LNF8|I3LNF8_PIG tr|F1SGS9|F1SGS9_PIG tr|A0A287B1V1|A0A287B1V1_PIG tr|F1RVV7|F1RVV7_PIG tr|F1SK95|F1SK95_PIG tr|K7GR16|K7GR16_PIG tr|F1SJQ4|F1SJQ4_PIG tr|A0A287BAK2|A0A287BAK2_PIG tr|D3K5K2|D3K5K2_PIG tr|A0A286ZP69|A0A286ZP69_PIG tr|I3LH25|I3LH25_PIG tr|A0A287AV05|A0A287AV05_PIG sp|A1DWM3|MFSD6_PIG tr|F1RI46|F1RI46_PIG tr|A0A286ZP67|A0A286ZP67_PIG tr|F1RFD3|F1RFD3_PIG tr|A0A287AQX8|A0A287AQX8_PIG tr|I3LP02|I3LP02_PIG tr|K7GR17|K7GR17_PIG tr|F2Z5M2|F2Z5M2_PIG tr|A0A286ZWI2|A0A286ZWI2_PIG tr|F1S2A8|F1S2A8_PIG tr|F1RL66|F1RL66_PIG tr|Q8SQB6|Q8SQB6_PIG tr|A0A0B8RVE3|A0A0B8RVE3_PIG tr|F1SC80|F1SC80_PIG tr|A0A287AYH9|A0A287AYH9_PIG tr|A0A287B9S4|A0A287B9S4_PIG tr|A0A287B1T7|A0A287B1T7_PIG sp|Q06A97|SEGN_PIG tr|Q8SQ66|Q8SQ66_PIG tr|F2Z568|F2Z568_PIG tr|D6PW25|D6PW25_PIG sp|P62901|RL31_PIG tr|K7GNY9|K7GNY9_PIG sp|P05027|AT1B1_PIG tr|F1SII4|F1SII4_PIG tr|B0FWK6|B0FWK6_PIG tr|I3L804|I3L804_PIG tr|A0A286ZP98|A0A286ZP98_PIG tr|F1RS74|F1RS74_PIG tr|K9J6K8|K9J6K8_PIG tr|F1RSH4|F1RSH4_PIG tr|I3LPM6|I3LPM6_PIG tr|A0A0B8S080|A0A0B8S080_PIG tr|F1SGS7|F1SGS7_PIG tr|F2Z5H6|F2Z5H6_PIG tr|A0A287A4F2|A0A287A4F2_PIG tr|A0A287A1G6|A0A287A1G6_PIG tr|F1SKG6|F1SKG6_PIG tr|F1SBY8|F1SBY8_PIG tr|F1SH89|F1SH89_PIG tr|A0A287A579|A0A287A579_PIG tr|F1SVC3|F1SVC3_PIG tr|F1ST14|F1ST14_PIG tr|F1REW6|F1REW6_PIG tr|F1SR64|F1SR64_PIG tr|F1SN95|F1SN95_PIG tr|A0A287AGD7|A0A287AGD7_PIG tr|A0A287BE34|A0A287BE34_PIG tr|F1S6W7|F1S6W7_PIG tr|F1RZS1|F1RZS1_PIG tr|F1S0G2|F1S0G2_PIG sp|P50441|GATM_PIG tr|F1SLA7|F1SLA7_PIG tr|A0A287B773|A0A287B773_PIG tr|F1S2D0|F1S2D0_PIG tr|F1S9F3|F1S9F3_PIG tr|I3LK24|I3LK24_PIG tr|A0A287AKP6|A0A287AKP6_PIG tr|F1SAK7|F1SAK7_PIG tr|A0A287A6K2|A0A287A6K2_PIG tr|F1SDP3|F1SDP3_PIG tr|A0A287APD5|A0A287APD5_PIG tr|A0A286ZQW5|A0A286ZQW5_PIG tr|F1SHF6|F1SHF6_PIG tr|I3LNH3|I3LNH3_PIG tr|A0A287AMM6|A0A287AMM6_PIG tr|F1S8M9|F1S8M9_PIG tr|K7GKS8|K7GKS8_PIG tr|I3LB23|I3LB23_PIG tr|A0A286ZJR5|A0A286ZJR5_PIG tr|A0A287A5U1|A0A287A5U1_PIG tr|A0A287AGU2|A0A287AGU2_PIG tr|M3VH80|M3VH80_PIG tr|F1RK45|F1RK45_PIG tr|F1SDC7|F1SDC7_PIG tr|K7GLT8|K7GLT8_PIG tr|I3LQN4|I3LQN4_PIG tr|D7RK08|D7RK08_PIG sp|P62802|H4_PIG tr|F1SPP8|F1SPP8_PIG tr|A0A287B5X3|A0A287B5X3_PIG sp|Q9GMB0|RPN1_PIG tr|F1S1A8|F1S1A8_PIG tr|A0A287AN76|A0A287AN76_PIG tr|A0A287AH93|A0A287AH93_PIG tr|A0A0B8S054|A0A0B8S054_PIG tr|F1SH64|F1SH64_PIG tr|K9IW70|K9IW70_PIG tr|A0A287A310|A0A287A310_PIG tr|I3LLW7|I3LLW7_PIG tr|F2Z4Z4|F2Z4Z4_PIG tr|A0A287A1C8|A0A287A1C8_PIG tr|Q28956|Q28956_PIG tr|F1SQD9|F1SQD9_PIG tr|F1RVZ1|F1RVZ1_PIG tr|F1RQT7|F1RQT7_PIG tr|F1RJC5|F1RJC5_PIG tr|A0A287AR56|A0A287AR56_PIG tr|A0A286ZSD7|A0A286ZSD7_PIG tr|K7GNU1|K7GNU1_PIG tr|F1RM04|F1RM04_PIG tr|K7GPJ4|K7GPJ4_PIG tr|F1S842|F1S842_PIG tr|A0A287BSP1|A0A287BSP1_PIG tr|F1S0E1|F1S0E1_PIG tr|M3UZ49|M3UZ49_PIG tr|F1SAY9|F1SAY9_PIG tr|A0A286ZUZ5|A0A286ZUZ5_PIG tr|F1RI32|F1RI32_PIG tr|F1RPB0|F1RPB0_PIG tr|A0A287AN37|A0A287AN37_PIG tr|F1S535|F1S535_PIG tr|K9IVU5|K9IVU5_PIG tr|F1SMF6|F1SMF6_PIG tr|K7GQ83|K7GQ83_PIG tr|Q5MJE5|Q5MJE5_PIG tr|A0A287ALB2|A0A287ALB2_PIG sp|O02670|KCNJ5_PIG tr|A0A287APR9|A0A287APR9_PIG tr|F1SCR9|F1SCR9_PIG tr|B2D2K7|B2D2K7_PIG 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tr|F1SG21|F1SG21_PIG tr|I3LFR2|I3LFR2_PIG tr|F1SG95|F1SG95_PIG tr|F1S569|F1S569_PIG tr|I3LQS7|I3LQS7_PIG tr|A0A287AB32|A0A287AB32_PIG tr|F1S3K5|F1S3K5_PIG tr|A0A287BQM5|A0A287BQM5_PIG tr|I3LN44|I3LN44_PIG tr|A0A287A9I6|A0A287A9I6_PIG tr|I3LLV0|I3LLV0_PIG tr|A0A287ALQ1|A0A287ALQ1_PIG tr|A0A287ALE4|A0A287ALE4_PIG tr|F1SRN4|F1SRN4_PIG tr|A0A286ZZ99|A0A286ZZ99_PIG tr|M3V815|M3V815_PIG tr|I3LGC6|I3LGC6_PIG tr|F1SNG6|F1SNG6_PIG tr|I3LQ10|I3LQ10_PIG tr|F1SH97|F1SH97_PIG sp|Q5D144|TFAM_PIG tr|F1RZZ1|F1RZZ1_PIG tr|F1SLR6|F1SLR6_PIG tr|A0A286ZIA2|A0A286ZIA2_PIG tr|D2D0E6|D2D0E6_PIG tr|F1RP44|F1RP44_PIG tr|F1RM47|F1RM47_PIG tr|F1S8V9|F1S8V9_PIG tr|A0A287B020|A0A287B020_PIG tr|F1RJM8|F1RJM8_PIG tr|K7GND3|K7GND3_PIG tr|K7GS49|K7GS49_PIG tr|F1RGS2|F1RGS2_PIG tr|K7GP28|K7GP28_PIG tr|F1S369|F1S369_PIG tr|A7KZR2|A7KZR2_PIG tr|A0A287AY02|A0A287AY02_PIG tr|K9J4P3|K9J4P3_PIG tr|A0A0B8RVZ5|A0A0B8RVZ5_PIG sp|Q29092|ENPL_PIG tr|A0A286ZVS0|A0A286ZVS0_PIG tr|I3L9N5|I3L9N5_PIG tr|A0A287A1H3|A0A287A1H3_PIG tr|F1SKN5|F1SKN5_PIG sp|Q0Z8U2|RS3_PIG tr|F1RSM2|F1RSM2_PIG tr|F2Z5I7|F2Z5I7_PIG tr|F1RGP6|F1RGP6_PIG tr|F1RTB6|F1RTB6_PIG tr|F1S031|F1S031_PIG tr|A0A286ZQ12|A0A286ZQ12_PIG tr|F1RX64|F1RX64_PIG tr|A0A287BGW3|A0A287BGW3_PIG tr|A0A2C9F348|A0A2C9F348_PIG tr|F1RGJ2|F1RGJ2_PIG tr|A0A286ZYE7|A0A286ZYE7_PIG tr|A0A0B8RTQ8|A0A0B8RTQ8_PIG tr|A0A287AR59|A0A287AR59_PIG tr|F1RZC3|F1RZC3_PIG tr|A0A287B340|A0A287B340_PIG tr|F1SDM6|F1SDM6_PIG tr|F1SNE5|F1SNE5_PIG tr|F1SS16|F1SS16_PIG tr|A0A287AP23|A0A287AP23_PIG tr|F1RHF0|F1RHF0_PIG tr|A0A287BNZ1|A0A287BNZ1_PIG tr|A0A287AA99|A0A287AA99_PIG tr|F1RIS8|F1RIS8_PIG tr|A0A287AL55|A0A287AL55_PIG tr|F1RGD9|F1RGD9_PIG tr|I3L677|I3L677_PIG tr|I3LIN5|I3LIN5_PIG tr|A0A287BMK7|A0A287BMK7_PIG tr|A0A286ZI19|A0A286ZI19_PIG tr|A0A287A9D3|A0A287A9D3_PIG tr|A0A287AZX9|A0A287AZX9_PIG tr|F1SPR4|F1SPR4_PIG tr|A0A287BPJ1|A0A287BPJ1_PIG tr|A0A287BR94|A0A287BR94_PIG tr|A0A286ZQ28|A0A286ZQ28_PIG tr|A0A287AII8|A0A287AII8_PIG tr|F1SC51|F1SC51_PIG tr|K9J6K4|K9J6K4_PIG tr|F1RQP0|F1RQP0_PIG tr|A0A287B2P1|A0A287B2P1_PIG tr|F1S880|F1S880_PIG tr|A0A286ZPE3|A0A286ZPE3_PIG tr|A0A287AM26|A0A287AM26_PIG tr|A0A0K1TQQ7|A0A0K1TQQ7_PIG tr|F1RKY8|F1RKY8_PIG sp|Q52NJ4|ARL3_PIG tr|F1SP18|F1SP18_PIG tr|I3L929|I3L929_PIG tr|Q2TJA5|Q2TJA5_PIG tr|K9IVR7|K9IVR7_PIG tr|F1SB42|F1SB42_PIG tr|I3LQH7|I3LQH7_PIG tr|F1SGZ8|F1SGZ8_PIG tr|A0A286ZY37|A0A286ZY37_PIG tr|A0A2C9F397|A0A2C9F397_PIG tr|I3LQS2|I3LQS2_PIG tr|F1ST81|F1ST81_PIG tr|I3LDY2|I3LDY2_PIG tr|A0A287B9U5|A0A287B9U5_PIG tr|F1SJV8|F1SJV8_PIG tr|I3LKM9|I3LKM9_PIG tr|I3LDU2|I3LDU2_PIG tr|I3LBR7|I3LBR7_PIG tr|A0A287AUQ9|A0A287AUQ9_PIG tr|F1SI09|F1SI09_PIG tr|F1S325|F1S325_PIG tr|F1RSZ8|F1RSZ8_PIG tr|A0A288CG38|A0A288CG38_PIG tr|F1SS26|F1SS26_PIG tr|I3LF95|I3LF95_PIG tr|A0A287A018|A0A287A018_PIG tr|A0A287BAM0|A0A287BAM0_PIG tr|A0A287AKU6|A0A287AKU6_PIG tr|K7GN83|K7GN83_PIG tr|F1S2E2|F1S2E2_PIG tr|F1RT62|F1RT62_PIG tr|I3LEC2|I3LEC2_PIG tr|I3L5X7|I3L5X7_PIG tr|F1RKI3|F1RKI3_PIG tr|D0G777|D0G777_PIG tr|C0LZL0|C0LZL0_PIG sp|Q9TSX9|PRDX6_PIG sp|P10775|RINI_PIG tr|B6DX82|B6DX82_PIG tr|A0A287B975|A0A287B975_PIG tr|F1SQ01|F1SQ01_PIG tr|A0A287B781|A0A287B781_PIG tr|F1S1X9|F1S1X9_PIG tr|F1RPV1|F1RPV1_PIG tr|A0A287AYZ7|A0A287AYZ7_PIG tr|D6BV38|D6BV38_PIG tr|F1RPX2|F1RPX2_PIG tr|A0A287BEJ3|A0A287BEJ3_PIG tr|A0A287AEH0|A0A287AEH0_PIG tr|F1S0P4|F1S0P4_PIG tr|G9BFT9|G9BFT9_PIG tr|F1SGN8|F1SGN8_PIG tr|M3TYC8|M3TYC8_PIG tr|A0A287B356|A0A287B356_PIG tr|F1S1X3|F1S1X3_PIG tr|I3LPL0|I3LPL0_PIG tr|F1RM03|F1RM03_PIG tr|F1S0M3|F1S0M3_PIG tr|A0A287AU26|A0A287AU26_PIG tr|A0A287AAC3|A0A287AAC3_PIG tr|A0A0B8RVL1|A0A0B8RVL1_PIG tr|B6EAV5|B6EAV5_PIG tr|F1RKS3|F1RKS3_PIG tr|F1RIP4|F1RIP4_PIG tr|F1SMW4|F1SMW4_PIG tr|F1S431|F1S431_PIG tr|I3LQ79|I3LQ79_PIG tr|F1S8G6|F1S8G6_PIG tr|A0A287ADA9|A0A287ADA9_PIG tr|I3LB68|I3LB68_PIG tr|F1RSN3|F1RSN3_PIG tr|F1SFH7|F1SFH7_PIG tr|F1S131|F1S131_PIG tr|F1SP93|F1SP93_PIG tr|M3VH94|M3VH94_PIG tr|F1S2V7|F1S2V7_PIG tr|A0A287BDJ1|A0A287BDJ1_PIG tr|I3LVF5|I3LVF5_PIG tr|F1SFF8|F1SFF8_PIG tr|F1SIJ9|F1SIJ9_PIG tr|F1SUZ5|F1SUZ5_PIG tr|A0A287BDU1|A0A287BDU1_PIG tr|A0A287AJL0|A0A287AJL0_PIG tr|Q1W2G0|Q1W2G0_PIG tr|A5A8V6|A5A8V6_PIG tr|I3LHB2|I3LHB2_PIG tr|F1SBI1|F1SBI1_PIG tr|F1SA98|F1SA98_PIG tr|F1S9Q1|F1S9Q1_PIG tr|A0A287A2R9|A0A287A2R9_PIG tr|F2Z558|F2Z558_PIG tr|F1SDR7|F1SDR7_PIG tr|F1RIF8|F1RIF8_PIG tr|A0A0B8S0B1|A0A0B8S0B1_PIG tr|F1RVY7|F1RVY7_PIG tr|F1SDJ8|F1SDJ8_PIG tr|F1S5N0|F1S5N0_PIG tr|I3L813|I3L813_PIG tr|K9IVP8|K9IVP8_PIG tr|Q29057|Q29057_PIG tr|F1RYY5|F1RYY5_PIG tr|B5APU8|B5APU8_PIG tr|B6VAP9|B6VAP9_PIG tr|A0A287B4J2|A0A287B4J2_PIG tr|F1SIV3|F1SIV3_PIG tr|K7GLP9|K7GLP9_PIG tr|A0A287AVN7|A0A287AVN7_PIG tr|F1SE06|F1SE06_PIG tr|F1STL2|F1STL2_PIG tr|I3LKZ2|I3LKZ2_PIG tr|F1S3E8|F1S3E8_PIG tr|A0A287AC67|A0A287AC67_PIG tr|F1SQN4|F1SQN4_PIG tr|F1SQR0|F1SQR0_PIG tr|F1SMG8|F1SMG8_PIG tr|B5APU6|B5APU6_PIG tr|F2Z521|F2Z521_PIG tr|D2KPI8|D2KPI8_PIG tr|A0A287BDZ6|A0A287BDZ6_PIG tr|A0A287AH69|A0A287AH69_PIG tr|I3LUD1|I3LUD1_PIG tr|A0A287B3X9|A0A287B3X9_PIG tr|F1SUG3|F1SUG3_PIG tr|I3LDA5|I3LDA5_PIG tr|I3LBB2|I3LBB2_PIG tr|A0A287A3N7|A0A287A3N7_PIG tr|F1S475|F1S475_PIG tr|F1S833|F1S833_PIG tr|A0A287ALM0|A0A287ALM0_PIG tr|F1SD63|F1SD63_PIG tr|A0A287AZ21|A0A287AZ21_PIG tr|F1S2L2|F1S2L2_PIG tr|A0A287AUH4|A0A287AUH4_PIG tr|F1S0A3|F1S0A3_PIG tr|A0A287B0Q6|A0A287B0Q6_PIG tr|A0A287ALN1|A0A287ALN1_PIG tr|I3L770|I3L770_PIG tr|F1RVS1|F1RVS1_PIG tr|M3V7Z1|M3V7Z1_PIG tr|A0A287ACQ4|A0A287ACQ4_PIG tr|A0A287AXH8|A0A287AXH8_PIG tr|M3V819|M3V819_PIG tr|F1SU03|F1SU03_PIG tr|A0A287BG14|A0A287BG14_PIG tr|I3LBD0|I3LBD0_PIG tr|A0A286ZXJ1|A0A286ZXJ1_PIG tr|A0A287BSQ1|A0A287BSQ1_PIG tr|F1RN46|F1RN46_PIG tr|F1RYJ5|F1RYJ5_PIG tr|F6Q5P0|F6Q5P0_PIG tr|F1RMT5|F1RMT5_PIG tr|G9F6X8|G9F6X8_PIG tr|F1SA74|F1SA74_PIG tr|K9IW91|K9IW91_PIG tr|F1RG16|F1RG16_PIG sp|P62197|PRS8_PIG tr|I3LLT2|I3LLT2_PIG tr|F1RWC5|F1RWC5_PIG tr|A0A0B8RW53|A0A0B8RW53_PIG tr|A0A286ZJM4|A0A286ZJM4_PIG tr|I3L7E5|I3L7E5_PIG tr|A0A287BNE9|A0A287BNE9_PIG tr|F1SBM2|F1SBM2_PIG tr|F1SB63|F1SB63_PIG tr|I3LGD4|I3LGD4_PIG tr|A0A287AU47|A0A287AU47_PIG tr|F1SGF1|F1SGF1_PIG tr|Q6QAS5|Q6QAS5_PIG tr|F6Q1M2|F6Q1M2_PIG tr|A0A287AYY7|A0A287AYY7_PIG tr|B9TSR5|B9TSR5_PIG tr|D0G0C8|D0G0C8_PIG tr|I3LJ87|I3LJ87_PIG tr|F1RR89|F1RR89_PIG tr|F1SKI5|F1SKI5_PIG tr|F1S0R7|F1S0R7_PIG tr|A0A286ZUW0|A0A286ZUW0_PIG tr|M3TYC1|M3TYC1_PIG tr|A0A287BM79|A0A287BM79_PIG sp|P63246|RACK1_PIG tr|I3LVK3|I3LVK3_PIG tr|A0A286ZRM3|A0A286ZRM3_PIG tr|A0A287ABK2|A0A287ABK2_PIG tr|I3LL84|I3LL84_PIG tr|I3LLA8|I3LLA8_PIG tr|A0A2C9F366|A0A2C9F366_PIG tr|F1RQ91|F1RQ91_PIG tr|I3L9C8|I3L9C8_PIG tr|A0A287AL76|A0A287AL76_PIG tr|F1RRK9|F1RRK9_PIG tr|I3LKK0|I3LKK0_PIG tr|I3L9J4|I3L9J4_PIG tr|M3UZ46|M3UZ46_PIG sp|A4Z6H1|PURA2_PIG tr|A0A286ZZP8|A0A286ZZP8_PIG tr|F1SML4|F1SML4_PIG tr|D0G6S0|D0G6S0_PIG tr|F1RRN1|F1RRN1_PIG tr|F1RZA6|F1RZA6_PIG tr|A0A287AJJ2|A0A287AJJ2_PIG tr|K9IVR2|K9IVR2_PIG tr|F1S8G0|F1S8G0_PIG tr|A0A287AKY8|A0A287AKY8_PIG tr|F6Q330|F6Q330_PIG tr|F1SAB2|F1SAB2_PIG bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint tr|I3LC63|I3LC63_PIG (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. tr|A0A287AG36|A0A287AG36_PIG tr|F1RHZ9|F1RHZ9_PIG tr|F1RLL9|F1RLL9_PIG tr|I3LR86|I3LR86_PIG tr|F1SPF3|F1SPF3_PIG tr|A0A287BKE1|A0A287BKE1_PIG tr|F1RSE7|F1RSE7_PIG tr|I3LM93|I3LM93_PIG sp|P20112|SPRC_PIG sp|P06867|PLMN_PIG tr|F1S663|F1S663_PIG tr|F1RHG1|F1RHG1_PIG tr|I3LMH3|I3LMH3_PIG tr|F1SVA2|F1SVA2_PIG tr|F1SQN1|F1SQN1_PIG tr|F1SMW9|F1SMW9_PIG tr|A0A287B1I4|A0A287B1I4_PIG tr|A0A287BP37|A0A287BP37_PIG tr|I3LLD5|I3LLD5_PIG tr|E1CAJ5|E1CAJ5_PIG tr|A0A286ZP03|A0A286ZP03_PIG tr|A0A286ZIH0|A0A286ZIH0_PIG sp|P28491|CALR_PIG tr|F2Z5D2|F2Z5D2_PIG tr|I3LMH4|I3LMH4_PIG sp|Q19PY3|RTCB_PIG tr|A0A287AN02|A0A287AN02_PIG tr|A0A286ZLI8|A0A286ZLI8_PIG tr|F1S982|F1S982_PIG tr|F1SAD7|F1SAD7_PIG tr|A0A287B1T2|A0A287B1T2_PIG tr|A0A173G6G8|A0A173G6G8_PIG sp|Q8MIZ3|DCPS_PIG tr|F1SL99|F1SL99_PIG tr|F2Z509|F2Z509_PIG tr|B3VMR0|B3VMR0_PIG sp|O02705|HS90A_PIG tr|A0A0B8RZM8|A0A0B8RZM8_PIG tr|F1RT25|F1RT25_PIG sp|Q2QLE3|TES_PIG tr|K7GRY0|K7GRY0_PIG tr|A0A287AWI9|A0A287AWI9_PIG tr|A0A287A4Q7|A0A287A4Q7_PIG tr|A0A287A699|A0A287A699_PIG tr|F1SLU6|F1SLU6_PIG tr|M3VK13|M3VK13_PIG tr|I3LCA2|I3LCA2_PIG tr|A0A286ZXU2|A0A286ZXU2_PIG tr|F1SMV6|F1SMV6_PIG tr|D0G7F7|D0G7F7_PIG tr|F1RYY0|F1RYY0_PIG tr|Q0PY11|Q0PY11_PIG tr|F1RPW9|F1RPW9_PIG tr|Q6QA25|Q6QA25_PIG sp|P54612|2AAA_PIG tr|I3LNK5|I3LNK5_PIG tr|A8HR17|A8HR17_PIG tr|A0A2C9F331|A0A2C9F331_PIG tr|F1SJR7|F1SJR7_PIG tr|F2Z5Q3|F2Z5Q3_PIG tr|F1RMJ9|F1RMJ9_PIG tr|A0A287A9T4|A0A287A9T4_PIG tr|I3LP37|I3LP37_PIG tr|O62708|O62708_PIG tr|A0A287AZG0|A0A287AZG0_PIG tr|K9IWF9|K9IWF9_PIG tr|D0G0C9|D0G0C9_PIG tr|A0A287A5B3|A0A287A5B3_PIG tr|F1RS49|F1RS49_PIG tr|F1RRD3|F1RRD3_PIG tr|K9IVP5|K9IVP5_PIG tr|A0A287B9W3|A0A287B9W3_PIG tr|A0A287AR84|A0A287AR84_PIG tr|A0A287AMZ2|A0A287AMZ2_PIG tr|I3LLY3|I3LLY3_PIG tr|A0A287AKI9|A0A287AKI9_PIG sp|P62272|RS18_PIG tr|A0A287BAK1|A0A287BAK1_PIG bioRxiv preprint doi: https://doi.org/10.1101/2020.02.24.962738; this version posted February 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure S-2 | The cluster data of all identified differential proteins in 8 groups. The
cluster analysis of identified proteins in 8 groups, of which the ordinate indicated the
near and far relationships according unique peptide of different identified proteins and
the abscissa indicated proximity of different groups. Green color represented proteins
down-regulated, red color represented proteins up-regulated and the color shade
degree indicated the degree of up and down regulated. Simultaneously, in the right of
the picture was listing the accession number of all these proteins in UniProt database.