Preparation Of ETEC K99 Gene Engineering Lactobacillus And Detection Of Protective Immunity

Enterotoxigenic Escherichia coli (ETEC) is one of the pathogens which can cause human and young livestock (nascent swine, calves, lambs, weaning swine) diarrhea. The morbility and mortality is very high in China even in the world wide, so it is very harmful to animal husbandry and cause enormous economic loss. The pathogenesis of ETEC is closely related to adhesions and enterotoxins. So far, the discovered adhesions of young stock ETEC have K88, K99, 987P, F41, F42, F165, F17, F18 and so on. Of those, K88, K99, 987P, and F41 are the most prevalent. In this study, Lactobacillus casei (L.casei) was selected as receptor strain to express K99 gene, which combined physiological function with specific immunity together. The experimental aim is to establish an antigen delivery system for the development of K99 oral vaccine.In this study, K99 gene of ETEC was cloned into expressing vector with Amp and Chl double labellings, which can shuttle and express in E.coli and L.casei. We have developed a surface antigen display system using pgsA (poly-γ-glutamate synthetase A) as an anchoring matrix. The recombinant fusion proteins comprised of pgsA and fimbriae protein of K99 were stably expressed on L.casei. The recombinant vector pLA-K99 was transformed into the competent cells L.casei. Western blotting analysis with rabbit-anti-K99 polyclonal serum indicated that the recombinant protein reacted with the specific antibodies. The results showed that the molecular weight of the recombinant protein was about 59kDa. The K99 fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. In addition, the survival of recombinant L.casei was studied in imitative gastrointestinal environments.The results indicated that the recombinant strain survived well in artificial gastric fluids, artificial intestinal fluid and 0.3%bile.Oral and intranasal immunization of SPF BALB/c mice was performed with the recombinant strain L.casei harboring pLA-K99 or pLA. Specific anti-K99 IgG antibody and IgG subclasses (IgG1, IgG2a, IgG2b) in the serum and specific anti-K99 secret immunoglobulin A (sIgA) antibody in the lung, intestines, vagina fluid and feces of mice were detected by indirect ELISA. Ten weeks after immunization with the recombinant L.casei, cytokine-specific analysis were detected by ELISPOT.The mice orally or intranasally immunized with pLA-K99/L.casei and pLA/L.casei were challenged with standard-type ETEC (C83912). The results showed significant sIgA titers that remained elevated for >16 weeks. High levels of IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b titers. Cytokine-specific ELISPOT showed induction of predominant Th2-type responses in both mucosal and systemic immune compartments supporting the IgG1 and IgA anti-K99 antibodies (Abs). More than 90% survived in oral immunization group whereas more than 85% survived in intranasal immunization group after challenged with C83912, while all of the mice dead in the control group. The recombinant pLA-K99/L.casei we have may become candidate oral vaccine for preventing calves from ETEC K99 infection.