Immune Response And Protective Research In Mice Induced By Immunization With Recombinant Lactobacillus Casei Expressing ETEC F41

Enterotoxigenic Escherichia coli(ETEC)is one of the pathogens which cause human and young livestock (nascent swine、calves、lambs、weaning swine) diarrhea. The morbility and mortality is very high in China even in the whole world,so it is very harmful to animal husbandry and cause enormous economic loss.In this study, Lactobacillus casei (L.casei) was selected as receptor strain to express F41 gene, which combined physiological function with specific immunity together.The experiment was to establish an important material bases for the development of F41 oral vaccine.In an effort to develop a safe and effective vaccine for the prevention of ETEC F41 infections, 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 F41 were stably expressed on L.casei.The recombinant vector pLA-F41 was transformed into the competent cells L.casei. Western blotting analysis with mouse-anti-F41 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 73kDa. The F41 fusion protein on the cell surface was confirmed by immunofluorescence mciroscopy 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-F41or pLA. For oral immunization, the mice were inoculated daily on days 0 to 4, 7 to 11, 21 to 25, and 49 to 53. A lighter schedule was used for nasal immunization (days 0 to 2, 7 to 9, 21and 49). Specific anti-F41 IgG antibody and IgG subclasses (IgG1, IgG2a, IgG2b) in the serum and specific anti-F41 secret immunoglobulin A (sIgA) antibody in the lung, intestines, vagina fluid and feces of mice were detected by indirect ELISA. Four weeks and ten weeks after immunization with the recombinant L.casei, cytokine-specific analysis was detected by ELISPOT. The mice orally or intranasally immunized with pLA-F41 /L. casei and pLA/L. casei were challenged with standard-type ETEC F41 (C83919).The results showed significant sIgA titers that remained elevated for >16 weeks. High levels of IgG responses in serum specific for F41 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-F41 antibodies (Abs). More than 90% survived in oral immunization group whereas more than 85% survived in intranasal immunization group after challenged with C83919, all dead in the control group. Ninety percent of the pups survived in oral immunization group whereas 80% survived in intranasal immunization group after challenged with C83919, but only a 5% survival rate for pups that were either immunized with a control pLA vector or unimmunized.This study is expected to lay the foundation for futher studies on gene engineering L.casei oral vaccine in their prevention of F41.