Cloning,Expression Of K88 Fimbrial Subunit FaeG Gene And Praparation Of Its Monoclonal Antibody

Escherichia coli is one of the most important pathogens in young animals, causing diarrhea, oedema disease or colisepticemia. Infection of enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea and death in preweaned pigs.The organism has two types of virulence factors including fimbrial adhensins and enterotoxins. Fimbriae are hair-like proteinaceous appendages, playing an essential role in the pathogenesis of E.coli infection by mediating adhesion to epithelial cells. When the adhesins on the surface of bacteria bind to corresponding acceptor on the surface of host s cells, bacteria are stably adhered to host s cells so that the bacteria can localize in local tissues and produce toxin or destroy tissues, leading to diarrhea.To facilitate investigation into adhesion mechanism of F4+E.coli, faeG gene of 0.8kb was amplified by high fidelity PCR from six stranins of F4+ E.coli with over 95% identical to that previously published F4 fimbrial gene by DNA sequencing. The PCR product was digested by restriction enzymes and then subcloned into the prokaryotic expression vector pGEX-6p-1. The GST-FaeG fusion protein of about 53KDa was expressed in BL21 E.coli after IPTG induction, which was revealed by SDS-PAGE and confirmed by Western- blotting.The fusion protein was purified by Glutathione Sephrose 4B column and used to immunize BALB/c mice. SP2/0 myeloma cells and spleen cells of the immunized mice were fused by PEG-1000. The hybridoma culture supematants were screened forFaeG-specific antibody production by indirect-ELISA with three positive clones established. The result of Western blotting showed that the three monoclonal antibodies reacted positively with the GST fusion protein and not with the GST carrier protein expressed in E. coli.