Edwardsiella tarda anti-idiotypic single chain antibody gene construction expression and identification of.docVIP

 PAGE \* MERGEFORMAT 16 Edwardsiella tarda anti-idiotypic single chain antibody gene construction expression and identification of Of: Qin Hong, Jin Xiao-Hang, HUANG Wei, Liu Yulin [Abstract] Objective Construction, expression and identification of Edwardsiella tarda anti-idiotypic single chain antibody genes. Methods Using RT PCR method for the secretion of Edwardsiella tarda from a unique monoclonal antibody hybridoma cells (1E11) in the VH and VL can be cloned variable region gene, and then splicing by overlap extension PCR method in the VH and VL variable regions between genes, the introduction of linker peptide (Gly4Ser) 3, In vitro construction of single-chain antibody gene was cloned into expression vector pET 28a expression in E. coli expression products were purified by SDS PAGE, Western blot and identified ELASA. Results Edwardsiella tarda anti-idiotypic scFv single chain antibody gene in BL21 (DE3) bacteria was expressed, the expressed product to form insoluble inclusion bodies, after the dissolution inclusion bodies, purified and refolded in vitro to obtain a high purity single-chain antibody fragments, recombinant proteins of molecular weight 27ku.ELASA analysis confirmed Edwardsiella tarda anti-idiotypic single chain antibody scFv genes with the same primary antibody, with more high antigen affinity. Conclusion The successful construction of the Edwardsiella tarda anti-idiotypic single chain antibody gene scFv, for further preparation of Edwardsiella tarda genetic engineering anti-idiotypic antibody vaccine basis. [Keywords:] Edwardsiella tarda, anti-idiotypic single chain antibody ABSTRACT: Objective To construct, express and identify the anti idiotypic antibody single chain variable fragment (scFv) against Edwardsiella tarda. Methods By using RT PCR method, the variable regions of the heavy and light chain of the anti idiotypic monoclonal antibody (mAb) 1E11 against Edwardsiella tarda were cloned and joined with a (Gly4Ser) 3 linker,