Cloning, Expression Of F5 Fimbril 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. F5+ETEC is one of the important pathogens caused piglet diarrhea disease, To F5 fimbrial studying in a deepgoing way has very strong theory and practical or immediate significance.To facilitate investigation into adhesion mechanism of F5+ETEC, 496bp was amplified by high fidelity PCR using the DNA template of strain .coli987. By measuring order and the array analysing the compare, obtained a nucleotide array and the standard F5 bacterial strain that self own announces, array consanguinity sex reaches 99.8%. The PCR product was digested by restriction enzymes and then subcloned nto the prokaryotic expression vector pET-30a. The fusion protein of about 23Ku was expressed after by IPTG induction, which was revealed by SDS-PAGE. Acquire the monoclonal antibody against the fusion protein, the fusion protein was used to immunize BALB/c mice. The hybridoma culture supernatants were creened for F5-specific antibody production by indirect-ELISA. In this research, one positive clone was established. The result of Western blotting showed that the one monoclonal antibodies reacted positively with the recombine protein. By detecting specificity ,the McAb didn't react with F4+ and F6+.