Colonization And Antibody Levels Of Lactobacillus Casei Labeled GFP In Mouse Intestine After Oral Vaccination

As many parasitic pathogens enter the body via mucosal surfaces, mucosa immunization was very important to protect organisms from infection of these pathogens. New vaccination strategies have been developed in order to avoid disadvantages associated with parenterally administeration vaccines. Oral vaccination applied on a large scale,is relatively inexpensive and frequently induces both local and systemic immune responses, resulting in an effective blocking the route of pathogens in entry and infection site. Lactic acid bacteria (LAB) are considered to be safe organisms and advocated for use as live antigen delivery vehicles to mucosal sites.In this study, Green Fluorescent Protein (GFP) was cloned into the expression vectors pLA and pLA-A, which can shuttle and express in E.coli and Lactobacillus casei (L.casei), and transformed into the host cells L.casei by electroporation to generate recombinant becteria pLA-GFP/ L.casei. and pLA-A-GFP/ L.casei. The recombinant fusion proteins were stable expressed on cell surface using the poly-r-glutamic acid synthetase A protein (pgsA) as an anchoring matrix. The molecular weight of the recombinant protein was about 69 kDa and 79 kDa in the result of Western blot. The GFP fusion protein on the cell surface was confirmed by fluorescence microscopy and flow cytometric analysis. We orally dosed up six-week-old female SPF BALB/c mice with the recombinant L.casei of approximately 109. Groups of at least three mice per condition were sacrificed at 1.5 h, 3 h, 12 h, 1 d, 3 d, 5 d, 6 d, 7 d, and its duodenum, jejunum, ileum, caecum intestinal tract rinse solution was sampled separately. The recombinant bacteria in intestinal tracts were examined by the method of plate culture count. The ratio of the seventh day to the first day of the recombinant L.casei adhered to the intestinal mucosa in the duodenum, jejunum, ileum, and caecum was 16.49%, 25.08%, 47.71%, and 41.03%, respectively. Our findings indicated that L.casei used as a deliver vector in oral vaccine is feasible.Oral immunization of SPF BALB/c mice was performed with the recombinant strain L.casei harboring pLA-A-GFP. Specific anti-TGEV IgG antibody in the serum and specific anti-TGEV secret immunoglobulin A (sIgA) antibody in the lung intestines fluid and feces of mice were detected by indirect ELISA. The results showed that the mice immunized with recombinant bacteria produced high level of anti-TEGV IgG in the serum and high level of sIgA antibodies were detected in the vaccinated group. The results of five consecutive days oral immunization group have higher level of antibody than others. These results indicated that the recombinant pLA-A-GFP/L.casei could induce serum IgG responses as well as specific mucosal IgA responses, and evoked both system immune and mucosal immune responses against TGEV.