The Construction Of The Sepa Gene Deletion Mutants Of K88ac~+ Enterotoxigenic Escherichia Coli And Related Function Analysis

Enterotoxigenic Escherichia coli (ETEC) is the main pathogenic bacteria which can cause significant watery diarrhea among young animals, especially in post-weaning piglets, and results in high morbidity and mortality, which bring huge economic loss. ETEC is characterized by the production of K88 adhesin that can mediate ETEC adherence to the host intestinal epithelium, then enterotoxins can interact with the intestine to cause watery diarrhea.Based on the original sequences of sepA gene in Genebank, specific primers were used to detect and amplify sepA gene in C83902 (Wild-type, O8:H87:H19). Plasmid pKD3 was used as template to generate PCR products which contain chloramphenicol resistance gene flanked by homology extensions of sepA gene. And then we transformed PCR products into C83902 with plasmid pKD46 by electroporation. The target gene was replaced by the cat gene, by X Red-mediated recombination system. The successful recombinant bacteria’s cat gene was eliminated in the second recombination process with the help of FLP recombinase expressed by pCP20. Finally, the C83902ΔsepA mutant was confirmed by PCR amplification and DNA sequencing.In adherence assay, ΔsepA mutants showed about 55.3% reduction in adherence to IPEC-J2 cells in vitro; the result of the realtime PCR test demonstrate that C83902AsepA has no significant effect on the transcription of fliC、faeG、eltB、hlyA、fimA; the rabbit intestinal loop assay indicated that fluid accumulation and the degree of damage of the ileal loop caused by AsepA mutants is much more serious than wild type strains.Above all, SepA plays an important role in colonization of C83902 strains to host cells. Then SepA may inhibit more expression of enterotoxin in strain C83902 in order to set up the balance between pathogens and host.