Construction Of Recombinant Lactobacillus Casei Coexpressing ETEC K99,K88 And Research Of Immunogenicity

Enterotoxigenic Escherichia coli (ETEC) is one of the major pathogens which can lead to diarrhea diseases among young nascent swine or weaning swine, causing great financial losses to animal husbandry in China even all over the world. The pathogenesis of ETEC is closely related to adhesions and enterotoxins. It is the prerequisite of diarrhea piglets that the adhesions is combined with specific receptors of the small intestinal epithelium cells and colonized in mucosa surface. So far, the discovered adhesions of young stock ETEC have K88, K99, 987P, F41 and so on, and K88, K99 of those are the most prevalent.In this study, a recombinant plasmid (pLA-K99-K88-LTB) harboring surface expression vector pLA and the ETEC K88, K99 gene was constructed and then transformed into Lactobacillus casei CICC6105 by electroporation, resulting in recombinant strain pLA-K99-K88- LTB/L.casei. The recombinant strains were induced to express interest protein, which was detected by Western blotting, immunofluorescence microscopy and flow cytometry. The results showed that a molecular weight of about 90 kDa was detected by Western blotting and the fusion protein expressing on the cell surface of L.casei using pgsA, a membrane-anchored protein display motif was confirmed by fluorescence microscopy and flow cytometric analysis.SPF C57BL/6 mice as an animal model were inoculated orally with recombinant strains pLA-K99-K88-LTB/L.casei, pLA-K99/L.casei and pLA-K88/L.casei. Specific anti-K88 or K99 IgG antibody in the serum and specific anti-K88 or K99 secret immunoglobulin A (sIgA) antibody in the lung, intestines, vagina fluid and feces of immunized mice were detected by indirect ELISA. T-cell proliferation responses were investigated by MTT test. The mice orally immunized with pLA-K99-K88-LTB/L.casei and pLA-K99/L.casei were challenged with standard-type C83912. The mice orally immunized with pLA-K99-K88-LTB/L.casei and pLA-K88/L.casei were challenged with standard-type C83902. The results of ELISA showed that the mice immunized with recombinant strains could produce remarkable specific sIgA level in the feces samples and lavage fluids samples, and high level of anti-K88 or K99 specific IgG in the serum. Significant difference was observed among the spleen lymphocyte proliferation index (LPI) among mice of the immunization groups and control groups. More than 80% of the vaccinated mice were protected against challenge with a lethal dose of standard stains C83912 and C83902. In addition, The results of ELISA demonstrated that the level of humoral immune response and cellular immune response in mice immunized with pLA-K88/L.casei and pLA-K99/L.casei are higher than the group immunized with pLA-K99-K88-LTB/L.casei.In conclusion, the oral immunizations with recombinant L.casei can induce significant specific mucosal sIgA response as well as serum IgG responses, and can evoke both mucosal immune and system immune responses.