Prokaryotic Expression, Purification, And Immunogenicity Analysis Of Etec Fasa Subunit

Enterotoxigenic Escherichia coli (ETEC) is one of the most important pathogens in severe watery diarrhea in piglets, calves, lambs and other farm livestock, its high morbidity and mortality caused big economic losses. Two protective antigens is closely related to its pathogenesis:enterotoxins and adhesins, that also named as pili. Epidemiological studies have shown the diversity of the serotypes of adhesins, the most common are K88、K99、987P、F41, and the987P is predominant.In this study, the gene encoding FasA subunit of the987P pili of987P+ETEC was cloned into a prokaryotic expressional vector, purified FasA subunit was used to analyse its immunogenieity.Part Ⅰ:fasA gene without signal peptide sequence of987P+ETEC was cloned into the prokaryotic expressional vector pQE-30, the recombinant plasmid pQE-30-fasA and transformed into E.coli M15for expression induced by IPTG (Isopropyl P-D-1-Thiogalactopyranoside). FasA subunit was isolated and purified by Ni-Agarose His-tagged protein purification kit, then be saved at-70℃after dialysis and renaturation. Part II:The purified and renatured FasA subunit was vaccinated BALB/c mice for preparing the specific antiserum. The titer of the antiserum was analyzed by ELISA.In the study we successfully constructed the expressional vector pQE-30-fasA and FasA subunit was highly expressed induced by IPTG. The titer of the antiserum was1:125000. It showed the high-performance immunogenicity of FasA subunit. The results may lay a foundation for developing novel Bifidobacterium-based oral vaccines against ETEC in future.