Research Of Recombinant Single Chain Variable Fragment Against Xanthomonas Axonopodis Pv. Citri

Recombination monoclonal antibodies (RMA) are artificial constructions produced by recombining the antibody molecule or synthesizing the gene sequence with gene recombination technology and then transforming the antibody DNA into cells for expression. The RMAs keep the binding properties of classical antibody. Among the RMAs, the single chain variable fragments (ScFvs) have been widely applicated to medical researches because of its reduced immunogenicity, small molecule, high tissue penetration, low production cost. ScFvs can be made by phage display or ribosome display technology, and the former have some obvious advantages than the later, such as simple method for construction of library and selection of antibody, no panning pressure, and improved antibody affinity by introducing mutation.There have been few reports about recombination monoclonal antibodies against phytopathogen except several reports about selection of ScFvs for diagnosing research by phage display. There have been no reports about selection of ScFvs against phytopathogen by ribosome display. Citrus canker caused by the bacterial pathogen Xanthomonas axonopodis pv. citri (Xac), a gram-negative bacterium, is a severe bacterial disease of most commercial citrus species and cultivars around the world, as well as some citrus relatives. The pathogen is the target of quarantine efforts abroad and domestics, then the development of rapid and reliable procedures for diagnosis and control of this pathogen has been an important priority. Lippolysaccharide (LPS) is a major component of outer membrane of gram-negative bacteria, and is suspected to be an important molecule for adhesion to and infection of plants. The LPS consists of O-specific side chains, core polysaccharide and lipid A, in which the O-specific side chain determines species specificity.For the study presented here, the O-specific LPS of Xac was used as an antigen to pan specific ScFvs against Xac by ribosome display. The selected ScFvs can be further applied to development of diagnostic reagent of Xac and research on the interaction between Xac pathogen and citrus during the phase of initial attachment-infection process. The main results are as follows:â‘ The Xac cell suspensions were used to immune the BALB/c mice and the titer of immunized mouse was 2500-fold. â‘¡The mRNA of mouse spleen cell was extracted for construction antibody library. The size of amplificated VH was 350 bp and that of VL was 650 bp. The VH and VL were ligated with the linker (Gly3Ser)4 and the assembled ScFvs were of expected 1.2 kb size. The ScFv DNA was cloned into PMD18-T, and nine clones were selected randomly for sequencing. The VH and Vk gene families of the ScFvs were designated based on Werner Muller's database (DNAPLOT software). The heavy chains of those belonged to the VH,VH2 and VH3 and the light chains belonged to the VKâ… , VKâ…¢and Vk IV subgroup, respectively. Each CDR of ScFvs was different and the amine acid sequences of the CDRs were diversity. It showed that the constructed library was diversity and could be used to selection of specific ScFvs.â‘¢In each round of ribosome display, the VH/k-DNA library was used for in vitro transcription with T7 RNA polymerase and the mRNA transcripts was translated in rabbit reticulocyte lysate system to produce antibody–ribosome–mRNA (ARM) complexes. The ARM complexes were then added to the O-specific LPS-coated microtiter plates and incubated. After washing, the retained ARM complexes were dissociated and the released mRNA was recovered by RT-PCR. The progress of panning was monitored by examining the intensity of RT-PCR products on agarose gel. By the end of the first round, only a weak DNA band was visible. The quantity of RT-PCR products continually increased during the next rounds of panning. Based on the result of RT-PCR, enrichment of specific ScFvs was clearly confirmed.â‘£DNA outputs from the unselected and the third selected antibody library were ligated with express vector pCANTAB-5E and then cloned into E.coli TG1 for soluble ScFvs expression. After transformation, 120 clones from the two libraries were isolated randomly and soluble proteins of these clones were expressed. The periplasmic extracts from these clones were tested for production of antigen-specific ScFvs by indirect ELISA. The result showed that all the isolated clones from the unselected library had little binding activity to O-specific LPS, but about 30% of the clones from the third selected library had a good conjugation activity. It also indicated that the ScFvs against O-specific LPS were enriched by ribosome display. Approximately 180 clones from the third selected library were analyzed by ELISA and about 60 clones of those reacted positively with O-specific LPS. Among them, 3 clones showed high affinity by Biacore analysis.⑤The selected antigen-positive ScFvs were transferred into HB2151 for high soluble expression. Briefly, 1ml overnight culture was added to 50 ml of freshly prepared 2×YT medium containing 100μg/ml ampicillin and 2% (w/v) glucose and cultured at 30℃with shaking at 250 r/min until they reached an absorbance of 0.6-0.8 at 600 nm. Then the cells were centrifuged and the supernatants were removed. The sediment cells were resuspended in 50 ml of freshly prepared 2×YT containing 100μg/ml ampicillin and 1 mM IPTG, and then incubated for 7 h at 30℃with shaking at 250 r/min. The cells were pelleted and resuspended in 0.5 ml ice-cold 1×TES buffer (0.2 M Tris–HCl [pH 8.0], 0.5 mM EDTA, 0.5 M sucrose) and 0.75 ml icecold 1/4×TES buffer. After incubation on ice for 30 min, the cells were pelleted by centrifugation at 10, 000 r/min for 10 min and the supernatant was retained as periplasmic extracts containing the soluble ScFvs.â‘¥The TCA-concentrated periplamic extract and purified ScFvs were run on a denaturing polyacrylamide gel and about 32kDa protein band was produced. The TCA-concentrated periplamic extract on the denaturing polyacrylamide gel were transferred to PVDF membrane for western blot analysis. A band at 32kDa was apparent.⑦The specificity of the three selected ScFvs (GX95, GX44 and GX13) was tested by indirect ELISA and Biacore analysis. The Xac and other bacteria (Xanthomonas. oryzae pv. oryzae, Xooc; Xanthomonas. campestris pv. campestri, Xcc; Xanthomonas. oryzae pv. oryzicola, Xoc; B. subtilis; E. coli and 10 saprophytic xanthomonads isolated from leaves of healthy citrus) were tested. The results showed that the three ScFvs had good activity with Xac, while no cross reaction with the other bacteria. The equilibrium constant (KA) determined by BIAcore analysis for GX13, GX44 and GX95 were 1.98×10¹⁰ M^-1, 1.89×10¹⁰ M^-1 and 3.43×10¹⁰ M^-1, respectively.⑧The three ScFv GX95, GX44 and GX13 were sequenced. Using the DNA sequences, the VH and Vk gene families of the ScFvs were designated based on Werner Muller's database (DNAPLOT software). The heavy chain of ScFv GX44 and GX13 belonged to the VH1 gene family and that of GX95 belonged to the VH3 gene family. The light chain of GX44 and GX13 belonged to the Vk IV subgroup and that of GX95 belonged to the Vk III subgroup. Sequencing alignment using the Vector NTI software showed that the VH of GX44 and GX13 shared 89.67% homology and the Vk of GX95 and GX13 shared 92.53% homology.