Evaluation Of Immune Effect Of Recombinant Bacillus Subtilis Expressing VP6 Protein Of Porcine Rotavirus

In order to develop a live carrier vaccine inducing gastrointestinal mucosal immunity to effectively prevent porcine rotavirus disease,this study used Bacillus subtilis as the delivery carrier to deliver immunogenic protein VP6 of porcine rotavirus orally to the gastrointestinal tract of mice.The immune effect was evaluated.In this study,VP6 protein was anchored on the surface of Bacillus subtilis spores by the surface display system.The recombinant strain BSp WBBO-cot BVP6 of spores display type Bacillus subtilis was successfully constructed.The recombinant strain BSpWBO-VP6 of Bacillus subtilis was successfully constructed by secretory expression system.In order to compare the immune effect of the oral vaccine of recombinant Bacillus subtilis,a recombinant Lactobacillus NZp NZ8149-VP6 was constructed and the immunological effect of its oral vaccinewas compared with the oral vaccine of bacillus subtilis.Mice were immunized with the constructed recombinant bacteria by gavage to stimulate the mucosa and cause specific immune response,and the immune effect of the vaccine was detected and evaluated.Specific research results are as follows:1.Cloning and expression of VP6 protein of porcine rotavirus and preparation of antiviral serumThe VP6 gene of porcine rotavirus was cloned and inserted into the polyclonal site of the expression plasmid p ET-28 a to result into the recombinant plasmid p ET-28 a VP6.The recombinant plasmid was transfer into the competent cells of Escherichia coli Rosetta through heat shock.After the recombinant strain was induced by 1m M IPTG,SDS-PAGE gel electrophoresis showed that the exogenous protein VP6 was expressed in the form of inclusion bodies.Subsequently,the positive recombinant strain was induced in large quantities,the VP6 protein was collected and purified,and the rabbit polyantiserum against porcine rotavirus was used as the primary antibody,and the purified VP6 antigen protein was identified by WB.Specific protein band was observed.It indicated that VP6 protein was successfully expressed and could react with specific antibody.Finally,the optimal working concentration of VP6 protein and serum was determined to be 2.5μg/m L and 1:80 by ELISA,which provided the material and test basis for the subsequent immune evaluation experiments in mice.2.Construction and identification of recombinant strains expressing porcine rotavirus VP6 proteinFirstly,the shuttle plasmid pWBO between Escherichia coli and Bacillus subtilis was constructed,and its promoter and signal peptide were removed by PCR to obtain the linear fragment of the plasmid p WBBO.Then,Bacillus subtilis nucleocapsid protein cot B and VP6 proteins were fused into the whole gene by fusion PCR.cot B-VP6 fusion gene was seam Lessly cloned into the plasmid p WBBO by homologous recombination,and sprouting display plasmid PWBBO-cot BVP6 was constructed.The recombinant sporophyte display plasmid was transferedinto the competent cells of Bacillus subtilis 168 by electrical shock,and the recombinant sporophyte display strain BSp WBBO-cot BVP6 was constructed.WB and IFA confirmed the successful expression of VP6 protein,which was fused and displayed on the surface of the sporophyte.VP6 gene was inserted into the polyclonal site of the shuttle plasmid pWBO to construct the secreted expression plasmid pWBO-VP6.The plasmid was transferred into Bacillus subtilis 168 competent cells by electrical shock to construct the secreted expression recombinant Bacillus subtilis strain BSpWBO-VP6,and WB detected the successful secretion and expression of VP6 protein.In order to better evaluate the immune effect of Bacillus subtilis expressing VP6 protein,a recombinant strain of lactic acid bacteria expressing VP6 protein was constructedto compared and study by a same immunization.The VP6 gene was inserted into the polyclonal sites of the secretory expression plasmid p NZ8149 of Lactococcus lactis to construct the secretory expression plasmid p NZ8149-VP6.The plasmid was transferred into the competent cells of Lactococcus lactis NZ3900 by electrical shock to construct the secretory expression recombinant Lactococcus lactis strain NZp NZ8149-VP6.VP6 protein was successfully expressed secretly through WB detection.3.Evaluation of immune effect of recombinant strains expressing VP6 protein of porcine rotavirus in miceIn order to evaluate the oral immunization effect of recombinant strains of BSp WBBO-cot BVP6,BSpWBO-VP6 and NZp NZ8149-VP6 expressing porcine rotavirus VP6,we inoculated 7 week-old SPF female BALB/c mice in each group with the recombinant strains,and then evaluated the immune level of the mice.ELISA was used to detect specific s Ig A antibody level in stool and intestinal mucosa,specific Ig G antibody level in sera.Spleen lymphocyte proliferation was detected by CCK8 test and the IFN-γ and IL-4 m RNA expression of spleen lymphocyte and intestinal mucosa cells were detected by fluorescence quantification.These data were used to evaluate the effects of recombinant bacteria on systemic immunity and intestinal mucosal immunity of mice.ELISA results showed that the serum Ig G antibody levels of recombinant BSp WBBO-cot BVP6,BSpWBO-VP6 and NZp NZ8149-VP6 groups were significantly increased compared with the control group after 28 days of intragastical immunization,and the secretion level of Ig A antibody in feces and intestinal mucosa was also significantly higher than that in the control group(p<0.0001).Antibody production peaked at 42 days after immunization,and then declined.CCK8 detection of splenic lymphocyte proliferation showed that,compared with the control group,all experimental groups could specifically induce splenic lymphocyte proliferation(p<0.01),and the proliferation index of bacterial solution group of BSpWBO-VP6 was the highest,but the proliferation difference among experimental groups was not significant.Quantitative fluorescence data analysis showed that the m RNA expression levels of IFN-γ and IL-4 in spleen lymphocytes and intestinal mucosal cells of mice were increased in all experimental groups(p<0.0001).The comprehensive analysis showed that the m RNA level of IFN-γ and IL-4 of the mice of Bacillus subtilis group was better than that of Lactococcus lactis group,and the difference was significant.In conclusion,specific humoral immunity and cellular immunity were induced in mice immunized with BSp WBBO-cot BVP6 spores,BSpWBO-VP6 bacterial solution and NZp NZ8149-VP6 bacterial solution.The immune effect of recombinant Bacillus subtilis was better than that of recombinant Lactococcus lactis.In this study,Bacillus subtilis was successfully used to secretely express VP6 protein of porcine rotavirus,and the fusedly expressed on the surface of its spores.The immune test of mice showed that it could better induce specific humoral immunity,mucosal immunity and cellular immunity.These data laid the foundation for further study of its oral vaccine.