Synthesis of a Functional Single-Chain Antibody Against Citrus Tristeza Closterovirus in Bacteria K. L. Manjunath, M. Hooker, H. R. Pappu, S. S. Pappu, C. A. Powell, M. Bar-Joseph, C. L. Niblett, and R. F. Lee ABSTRACT. A synthetic gene that encodes the antigen-binding regions of the monoclonal anti- body (MAb) 17Gl1, which is specific for citrus tristeza closterovirus (CTV), was constructed and expressed in Escherichia coli. lMAb 17Gll reacts with a broad spectrum of CTV isolates and rec- ognizes a surface epitope which is destroyed when treated with sodium dodecyl sulfate. The poly- merase chain reaction products from the cDNAs of the variable regions of heavy and light chains of the immunoglobulin leader sequence were linked by a synthetic peptide. This construct was cloned downstream of the pelB leader sequence in PET 22b vector and expressed in E. coli. The expressed protein, purified by affinity chromatography, was found to have antigen binding proper- ties similar to 17Gll. The single chain antibody gene construct will be used for transgenic expres- sion in plants to study its role in control of CTV. Key words. Bacterial expression, coat protein, sequence, plantibodies, monoclonal antibodies. Citrus tristeza closterovirus have been cloned and expressed in (CTV) is one of the most destructive heterologous systems like bacteria, diseases of citrus worldwide. Various yeasts and plants (1, 17). Even control measures for control and pre- though plants lack an immune sys- vention of CTV include quarantine, tem, production of a specific antigen- use of disease-free budwood, mild binding antibody may interfere with strain cross protection, tolerant the virus replication (14) and pre- scion-rootstock combinations, and vent disease. Single-chain antibody biological control of vectors (11).The (SCAB) proteins are novel recombi- virus is still spreading into new nant proteins consisting of the vari- areas, and continues to cause severe able regions of heavy (VH) and light losses throughout the world and chain (VL) molecules of immunoglo- warrants development of better con- bulins joined by a linker peptide (1). trol strategies. During the last 10 Compared to immunoglobulins, the years, various molecular biological SCAB proteins are much smaller in strategies have been developed for size and do not require elaborate the control of virus diseases (16). assembly in the cell. These proteins Pathogen-derived resistance were found to retain the same bind- involves expression of viral gene ing specificities of the parent MAbs sequences in plants and has been whose VH and VL sequences were found to be very effective (16). used (1,17). Both complete (3,5)and Expression of antibodies against engineered antibodies (9) have been viral genes in plants provides an expressed in plants. Attenuation of alternative approach for virus dis- viral symptoms by expression of ease control that avoids introduction both full-length and engineered sin- of pathogen genes into transformed gle-chain antibodies in plants has plants (14). been reported (14, 15). Here, we Immunoglobulins are key mole- report the bacterial expression of a cules in animal immune systems. CTV-specific SCAB gene with the Specific interaction of these mole- objective of studying its binding cules with the antigen is one of the properties and comparing it with the first of several complex processes in parent MAb. This is a necessary step the immune system. Antibody genes for its future utilization in plant Thirteenth IOCV Conference, 1996- Citrus Tristeza Virus 39 expression and assessment of its room temperature for 8 min followed activity against CTV. by quick freezing in liquid nitrogen. The reaction tubes were thawed, and MATERIALS AND METHODS the reverse transcription reaction was carried out at 42°C for 45 min in Selection of monoclonal anti- a 25 pl reaction mix containing 50 body. A panel of 11 MAbs (Table 1) mM Tris-HC1, pH 8.3, 50 mM KC1, was screened by using a double anti- 10 mM MgCl,, 0.5 mM spermidine, body sandwich indirect (DASI) 10 mM dithiothreitol, 0.5 mM each ELISA against 60 CTV isolates. of dATP, dCTP, dGTP and dTTP, These isolates were from nine coun- RNasin (20 u) and AMV reverse tries and represent a full range of transcriptase (10 u) (Promega). biological diversity among the CTV Construction of the single groups. Isolates inducing decline, chain Fv antibody (SCAB) gene. seedling yellows, grapefruit stem VH and VL regions were amplified pitting, sweet orange stem pitting by polymerase chain reaction (PCR) and mild isolates were included (4, of the above cDNAs using degener- 11).MAbs 1-7 were developed using ate consensus primers, a-d (Table 2). purified virus preparations of CTV Ten pl of each of the above cDNA isolates T36 and B185 as antigens templates was used in 100 pl reac- (6); MAbs 8-10 were developed tion mix containing 10 mM Tris-HC1, against B227 antigen (Manjunath pH 9.0, 50 mM KCI, 2.5 mM MgCl,, et al., unpublished data); and MAb 0.5 mM dNTPs, primers (100 pM F10 was kindly provided by Lochy each) and Taq polymerase (2.5 u). Batista (Instituto de Investiga- The reaction mixtures were incu- ciones de Citricos, La Habana, bated in an automatic thermocycler Cuba). Microtiter plates were coated for 35 cycles at 94°C for 1 min, 57°C with protein-A-sepharose purified for 1 min and 72°C for 1 min fol- immunoglobulin-G preparation of a lowed by a final incubation at 72°C polyclonal antibody, 1051 at 1 pglml. for 10 min. The PCR products Antigen extractions were used at obtained using VH and VL primers 1:20 dilution. Ascites of MAb F10 were then treated with the Klenow was used at 1:10,000 dilution. Hybri- enzyme (Promega), phosphorylated doma supernatants of all other with T4 polynucleotide kinase (US MAbs were used at 1:3 dilution. Biochemicals) and ligated to SmaI RNA extraction and cDNA digested pUC 118 phagemid vector synthesis. Mouse hybridoma cells as described earlier (8).Transforma- secreting 17Gll antibodies (6) were tion of E. coli (DH5), selection of used for cDNA synthesis. Total RNA recombinant colonies with gene was extracted from the cells by using sequences of VH (W118) and VL the ULTRASPECTMRNA isolation (VL1118) and plasmid purifications system (Biotex Laboratories, Inc. were conducted by using standard Houston, TX). First strand cDNA protocols (12). Double-stranded DNA was synthesized to the variable templates from these colonies were regions of heavy (VH) and light (VL) then sequenced using the Sequenase chains in separate reactions by version 2.0 sequencing kit (US Bio- using primers specific for the 3' end chemicals) or by a DuPont Genesis of the framework region 4 (FR4) of 2000 DNA Analysis system at the the variable domain (Table 2). Dena- DNA Sequencing Core Facility of the turation of RNA and primer anneal- Interdisciplinary Center for Biotecn- ing were done by mixing 10 pg of nology Research (ICBR) at the Uni- total RNA with 10 mM methyl mer- versity of Florida. Nucleic acid curic hydroxide and an appropriate sequences were analyzed using com- 3' end primer, and incubating at puter programs of the University of Thirteenth ZOCV Conference, 1996-Citrus Tristeza Virus Thirteenth IOCV Conference, 1996- Citrus Tristeza Virus 41 TABLE 2 PRIMERS USED FOR AMPLIFICATION OF VARIABLE REGIONS OF HEAVY AND LIGHT CHAINS OF MONOCLONAL ANTIBODY 17Gll AND CONSTRUCTION OF THE SINGLE CHAIN VARIABLE REGION ANTIBODY. RESTRICTION SITES, START AND STOP CODONS AND LINKER REGION SEQUENCES ARE SHOWN. HEAVY CHAIN VARIABLE REGION a. 5' end primer 5'-(C/G)AGGT(C/G)(A/C)A(A/C)A(A/G)CTGCAG(CGAGTCGG3' b. 3' end primer 5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCC3' LIGHT CHAIN (KAPPA) VARIABLE REGION c. 5' end primer 5'-GACATCGAGCTCACCCAGTCTCCA3' d. 3' end primer 5'-CCGTTTCAGCTCGAGCTTGGTCCC3' SINGLE CHAIN ANTIBODY HEAVY CHAIN EcoRI start e. 5' end primer ~'-AAGAATTCGAGCTCAGAAACC~GAGGT(C/G)(A/C)A(A/G)CTGCAGC/ G)AG 3' Sac1 f. 3' end primer 5'-TTCGGAGCCAGATCCTGAGGATTTACCCTCTGAGGAGACGGTGACCG- TGGT 3' linker LIGHT CHAIN g. 5' end primer 5'-TCAGGATCTGGCTCCGAATCCAAAGTCGACGACATCGAGCTCACCCA- G 3' linker KvnI &~p h. 3' end primer 5'-AAGGTACCGGATCCTCACCGTTTCAGCTCGAGCTTGGTC3' BamHI XhoI Wisconsin Genetics Computer periplasmic transport of the Group (UWGCG). expressed protein and a 6-histidine A second set of primers were tag at the carboxy-terminus. The designed for construction of the cloned gene was expressed in a T7 SCAB gene (Table 2, e-h) to include RNA polymerase-based expression appropriate restriction sites at the system (13) using E. coli, strain BL ends, an initiation codon and a 14 21 (DE3) pLysS, according to the amino acid long synthetic linker manufacturer's protocol. Briefly, the peptide, EGKSSGSGSESKVD (2) cells were grown at 37°C until an between VH and VL. The PCR strat- A6,,n, = 0.6 to 1.0 was reached. egy used for construction of the Expression of the cloned gene was SCAB gene is shown in Fig. 1. The induced by adding isopropyl N- SCAB gene was assembled by over- thiogalactopyranoside (IPTG) to a lap PCR of a mixture of re-amplified final concentration of 1 mM and con- PCR products of VH and VL genes tinuing incubation for another 3 hr using the 5' primer of VH and the 3' at 37°C. Bacterial expression was primer of VL. The SCAB gene was monitored by Western blotting then cloned into the EcoRI and using goat anti-mouse (Fab' specific) BamHI sites of the pUC 118 vector antibody conjugated to alkaline and sequenced as before. phosphatase (Sigma). The SCAB Expression and analysis of protein was purified from induced SCAB protein. The SCAB gene was cells using Ni-NTA resin from subcloned from pUC 118 into the Qiagen Inc. according to the manu- SacI and XhoI sites of the PET 22b facturer's instructions for purifica- vector (Novagen) leading to in-frame tion of native cytoplasmic proteins.