Construction Of Recombinant Attenuated Salmonella Carrying S Protein Antigen Epitopes Gene Of Porcine Epidemic Diarrhea Virus And Research In Its Immunogenicity

Since2006, Porcine Epidemic Diarrhea (PED) which persisted in several provinces in China caused significant economic loss because of the serious damage to the pig-raising industry. Due to the reasons such as the weak protective immunity of inactivated vaccine and unstable immune effect of vaccine because of the presence of wild mutants, PED vaccine used at present can’t prevent and control the prevalence of PED in different districts of China. Therefore, develop a new safety and high efficiency gene engineered vaccine has importance of actual meaning significance.In our study, two fragments (498～638aa,744～771aa) of the neutralizing epitope regions of S protein of PEDV SC/DY strain were cloned to construct the recombinant attenuated salmonella S.C500/pVAXl-SAB which immunized orally with mice to test whether they have good immunogenicity or not.1. Gene cloning of SA, SB fragment and construction of recombinant plasmidPrimers were designed and synthesized according to S gene sequence of Porcine epidemic diarrhea virus (PEDV) CV777strain in GenBank, PEDV-SC/DY total RNA was extracted as template, RT-PCR amplification of456bp and105bp target fragment SA and SB was conducted, SA and SB gene fragments were cloned to into pMD-19-T respectively to get pMD-SA and pMD-SB; SB fragment was inserted to pMD-SA to generate pMD-SAB by restriction enzyme digestion and connection, then SAB fusion fragment were subcloned into pET-32a (+) to get pET-SAB. New primers were designed and synthesized according to restriction enzyme sites of pVAX1, pMD-SA and pMD-SAB as templates, PCR amplification of446bp and534bp target fragment SA’and SAB’was conducted to construct pMD-SA’ and pMD-SAB’, then SA’ and SAB’ were subcloned into pVAXl to get pVAX1-SA and pVAX1-SAB, then two plasmids were transformed into S.C500to get S.C500/pVAX1-SA and S.C500/pVAX1-SAB.2. Prokaryotic and eukaryotic expression of SAB fusion genepET-SAB was chemically transformed Ecoli.Rosetta (DE3), then induced by IPTG, SAB fusion protein which molecular weight was about36.5kDa mostly existed in the form of inclusionbody was successfully expressed, Western-blot showed that SAB fusion protein had good reactogenicity; pVAX1-SA and pVAX1-SAB were transformed into Vero cell respectively, expression of recombinant plasmids was identified by indirect immunofluorscence assay showing green fluorescence which indicate expressed target proteins had good reactogenicity.3. Safety, stability of recombinant salmonella and its oral immunization of miceMice orally immuned by different doses of recombinant salmonella were all alive without showing clinical symptoms infected by salmonella, indicating that recombinant salmonella were on pathogenic for mice. Doses of1×107CFU/mL and1×108CFU/mL were relatively safe for mice; Continuous passage culture of S.C500/pVAX1, S.C500/pVAX1-SA, S.C500/pVAXl-SAB without kalamycin in LB broth, the stability of plasmid in three recombinant salmonellas was all above90%, but sharply declined since80generation; The dynamic state, growth and decline of S.C500/pVAXl, S.C500/pVAXl-SAB in organs of mice was found that bacteria were isolated from feces,liver and spleen at1,3and7day after immunization, furthermore, the number of bacteria isolated from feces was bigger than these isolated from liver and spleen, bacteria could not be isolated in these three organs two weeks after immunization, indicating that the bacteria were removed out of mice by immune system; The result of immunization:S.C500/pVAXl-SA, S.C500/pVAX1-SAB and pVAXl-SA, pVAX1-SAB both induced specific humoral immune response in mice immunized, furthermore, S.C500/pVAXl-SA and S.C500/pVAX1-SAB also induced specific intestinal mucosa immune response, S.C500/pVAXl-SAB induced a higher level of PEDV specific antibody compared with the rest of three groups with significant difference(p<0.05).In general, S.C500/pVAXl-SAB induced a higher level of humoral response than that induced by S.C500/pVAX1-SA, meanwhile, also induced a relatively balanced Thl/Th2cellular immune response.Conclusion:Recombinant attenuated salmonella of S.C500/pVAX1-SAB have good immunogenicity. Our research set the stage for developing a new orally genetic engineering live carrier vaccine.