Construction Of A Phage Display Library Of Repertoire Fab Antibody From Mouse Immunized With Xcc

Citrus canker caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc), 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. Most countries and areas worldwide which produce citrus have suffered from CBCD including Asia, Oceania, Malaysia, South America and North America. The host plants are barely put to death directly, but they will suffer defoliation, fruit-fall, vitality decline and lesion on the fruits which greatly degrades the quality and economic value of the fruits. 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.Recombinant monoclonal antibodies (including Fab, scFv, Fv, etc.) which have dual function of treatment and diagnosis, are a new diagnostic test for immunology detection. Using the principle of antigen-antibodie's specific binding; it can be used for human disease, plant disease diagnosis, treatment and prevention. Moreover, compared with polyclonal and monoclonal antibodies, recombinant monoclonal antibody has higher affinity and specificity, lower detection limit, easier to be expressed in prokaryotic expression system, and so on. 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. But it is also unstable and prone to aggregation, it is difficult to get high concentration antibody and restricts its actual application. Compared with single-chain antibody scFv, the Fab antibody fragments which composed of the antibody light chain and Fd regions of heavy chain, have better stability, express active antibody in E. coli directly and have simple preparation. To our knowledge, no information is currently available regarding anti-Xac Fab antibody. So In this study, we construct a phage display library of repertoire Fab antibody from mouse immunized with Xac. This research can provides new reagent for Xac diagnosis, and it can be used to research the recognition mechanisms of Xac to its host, it may provide a new method to biological contrl of Xac. The research content mainly includes:â‘ The Xac cell suspensions were used to immune the BALB/c mice and the titer of immunized mouse was 6400-fold.â‘¡The mRNA of mouse spleen cell was extracted for construction antibody library and transcribed reversely into cDNA. The genes of mouse Ig light chains (680bp) and heavy chains Fd region (680bp) were amplified by PCR. By sequence comparison, we confirme the amplification of the corresponding genes were the antibody further.â‘¢By Sac I and Xba I sites, the light chain genes were ligated into the vector pComb3x. Recombinant light chain library was successfully constructed, its size was 1×10⁶ and the cloning efficiences were 100%.â‘£By Xho I and Spe I sits, the antibody heavy chain Fd genes were ligated into recombinant light chain library. The size of the Fab library was 2×10⁶ and the recombination rate was 80% as indicated by PCR and endonuclease digestion analysis. After infection of helper phage VCSM13, The titer of the library was 2.4×10¹¹ pfu.