High Efficient Expression And Reconstruction Of Recombinant Antibody Of Xanthomonas Axonopodis Pv. Citri

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. For the lack of resistant cultivars and specific chemicals, the following measures have been adopted to control CBCD for a long time: enhanced construction of non-quarantine area and phytosanitary system against the spread of Xac, eradication and mass destruction methods dealing with infected plants. 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.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, which hold dual function between therapy and diagnosis. Using antigen-antibodies specific binding principle, it can be used for human disease, plant disease diagnosis, treatment and prevention. Single chain variable fragment (ScFv) is artificial construction small molecular antibody produced by genetic engineering, whose heavy chain variable region and light chain variable region are spliced together with a connecting peptide using genetic engineering methods. Because of its low or no immunogenicity, small molecular weight, strong organizations penetrating power, low cost, large-scale production, and other characteristics has been widely used in the medical field, which may be used to identify target pathogens and prevent plant diseases including XAC. But 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 high efficient expression and stability reconstruction of recombinant antibody for citrus bacterial canker disease.The purpose of this study is to pave a new way for immunization diagnosis and exploration of integrated control of citrus bacterial canker disease, which combines high yield recombinant single-chain antibody technology with colloidal gold, aiming at the existing citrus canker rapid detection technology system has not been perfect, and conventional detection methods for CBCD include PCR detection, phage detection, enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay (DIA) are all time-consuming, complex and less sensitive in a certain extent, which can not fulfill the requirements of quarantine adequately.The main content in this study are as follows: single-chain antibody fragments (ScFv 95) selected by ribosome display was amplified using an assembly of polymerase chain reaction (PCR), and a recombined plasmid pET30a(+)-XAC-ScFv was constructed by inserting the single chain Fv gene into bacterial expression vector pET30a(+). PET30a(+)-XAC-ScFv was transformed into Escherichia coli BL21 (DE3) and expressed which was induced by IPTG. Products were purified though Ni-NTA His Bind resin. The collected antibodies were refolded by gel filtration chromatography, and activity assaying process and the stability of the initial antibody transformation was done, which 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‘ Succeed in amplify single-chain antibody fragments for citrus bacterial canker disease and transformed into Escherichia coli BL21 (DE3) linking with vector pET30a(+) after digestion.â‘¡Antibody was expressed as inclusion bodies induced by IPTG. Inclusion extract was run on a denaturing polyacrylamide gel and about 32 kDa protein band was produced. Products purified though Ni-NTA His Bind resin on the denaturing polyacrylamide gel were transferred to PVDF membrane for western blot analysis. A band at 32 kDa was apparent.â‘¢The collected antibodies were refolded by gel filtration chromatography (Sephacryl-200HR), and activity was tested by Dot-blot. The results showed the functional ScFv was obtained through renaturation. The LPS of Xac and other bacteria (Xanthomonas. oryzae pv. oryzae, Xooc; Xanthomonas. campestris pv. campestri, Xcc; Xanthomonas. oryzae pv. oryzicola, Xoc) was extracted and diluted by HEPPES buffer (10 mmol/L HEPES, 100 mmol/L NaCL pH7.4), than was injected into HPA chip. The appetency of single-chain antibody was tested by Biacore evauation software. The Biacore analysis indicates that XAC-ScFv-95 showed significant affinity to LPS of Xac. The equilibrium constant (KA) determined by Biacore analysis for ScFv was 2.72×10⁷ M^-1.â‘£Cysteine was imported into VH and VL of ScFv gene successfully by overlap and extension PCR, which lays the foundation for follow-up stability reconstruction of recombinant antibody.