Immunogenicity Analysis In Mice Induced By Recombinant Lactobacillus Casei Expressing PEDV-S1 Epitope

Porcine epidemic diarrhea virus (PEDV) , a member of the Coronaviridae,causes an economically important disease in neonatal piglets, which characterized by severe enteritis with anorexia,vomiting,acute diarrhea,dehydration and significant mortality in swine .During the past 30 years ,in spite of preparation of inactivated and live attenuated vaccine, but PED cases were out in many swine-raising countries , incurring serious economic losses worldwide due to the death of neonatal piglets and weight loss of the infected pigs , It is necessary to desiging antiviral immunization strategy of PEDV.In order to produce a viable organism expressing antigens for cheap and safe,.the S1 epitopes of PEDV was amplified using the pQE30-PS1, The genes were subcloned into the expression vector pLA, and then transformed into L.casei by electroporation,resulting in recombinant strainPEDV-S1/L.casei .The recombinant strain constructed in this study was cultured in MRS medium to express recombinant protein.The recombinant protein which express on the cell surface detected by Westernblot and indirect immunofluoresecnce microscop and flow cytometry .Oral and intranasal immunization of mice was performed with the recombinant strain L.casei haboring pLA-PS1 .The immune protocol was administered on five consecutive days , days o to4。A booster Immunization was given ,on days 7 to 11 and a seeond booster was given on days 18 to 22, the last booster was given on days 28 to 32 .The mice were fed with 5×109 recombinant strains. A lighter schedule was used for nasal immunization (days 0 to 2, 7 to 9, 21 and 28) .As control,The mice was immunized with L.casei by oral route and nasal route .Specific anti-PEDV protein slgA was detected by indirect enzyme Iinked immunosorbent assay (ELISA) in the feces,lung wash fluid, intestines wash fluid of mice after oral and intranasal immunization, and specifi IgG was detected by indirect ELISA in the serum of immunized mice。The results of ELISA showed that the mice immunized with recombinant strains could produce remarkable special slgA level in the feces,lung wash fluid, intestines wash fluid of mice , and highest level of anti-PEDV protein IgG in the serum of mice .These result indicated that the recombinant strains could induce both mucosal immune and system immune responses. To better characterize antibody responses against PEDV-S protein fragments, the levels of antigen-specific IgG subclasses(IgG1, IgG2a ,IgG2b)and other antibody isotypes IgA and IgM were assessed by ELISA.Both intranasally and orally immunized mice developed S protein specific antibodies that were predominantly IgG2b with moderate levels of IgG2a and IgG1. The subclass ratio calculated as IgG1/(IgG2a+IgG2b) was less than 0.4 indicating a Th1 type response.