Development And Application Of Genetically Engineered Vaccine For Porcine Epidemic Diarrhea

Porcine epidemic diarrhea（PED）is one of the important diseases that endanger the healthy development of the pig industry.It has acute and highly contagious characteristics,causing severe economic losses to the pig industry.Vaccination is the safest and most effective way to prevent PED.The ideal vaccine can effectively prevent porcine epidemic diarrhea virus（PEDV）infection,significantly reduce the clinical symptoms and mortality of PEDV-infected herds,and improve pig growth performance.However,the traditional PED inactivated vaccines and PED attenuated vaccines are not very effective in immune protection.Therefore,the development and development of fast-acting,efficient and long-acting new PED genetic engineering vaccines have important practical significance.S protein is an important structural protein and immunogenic antigen of PEDV,carrying the main epitope of B lymphocyte antigens,and is considered to be the main target for the development of PED genetic engineering vaccines.Omp16 is an outer membrane protein of Brucella,which is not only important for bacterial growth and reproduction and maintaining its balance in the body,but also participates in the processes of bacterial adhesion,extracellular protein and polysaccharide secretion and biofilm formation.Immune adjuvant activity can be used as a mucosal vaccine adjuvant for the development of new genetic engineering vaccines.In this study,the synthetic plasmid p UC57-Omp16-S_493-708 was used as the material,the fusion gene Omp16-S_493-708 was cloned,the recombinant lactic acid bacteria pVE5523-Omp16-S_493-708/ATCC393 was constructed,and the immune effect of the mouse was tested by immunoassay.The results showed that the recombinant lactic acid bacteria pVE5523-Omp16-S_493-708/ATCC393 carrying the Omp16-S_493-708 gene was successfully constructed.The recombinant lactic acid bacteria can effectively present the foreign genes carried by oral immunization of mice,and stimulate the immunized mice to produce specific Sexual serum antibodies and induced cellular immune responses in mice provide new ideas for the development of new PED genetic engineering vaccines in the future.The main research contents are as follows:1.Construction of PEDV S_493-708 and Omp16 dual gene prokaryotic expression vectorsIn this study,the synthetic plasmid p UC57-Omp16-S_493-708 was used as a template.The upstream primer F1 and downstream primer R1 of Omp16-S_493-708 were designed to amplify.The upstream primer F2 and downstream primer R2 of S_493-708 were amplified.The PCR was amplified separately.1134 bp and 660 bp target fragments Omp16-S_493-708 and S_493-708 were cloned,and Omp16-S_493-708 and S_493-708 were cloned into the E.coli expression vector p ET-28a（+）,respectively,and then transformed into BL21（DE3）Competent cells were obtained from p ET28a-Omp16-S_493-708/BL21（DE3）and p ET28a-S_493-708/BL21（DE3）recombinant bacteria.SDS-PAGE results showed that Omp16-S_493-708/BL21（DE3）and After being induced by IPTG,p ET28a-S_493-708/BL21（DE3）can express the target protein of Omp16-S_493-708 and 23KD of S_493-708 with a size of about 40KD.The target protein is more concentrated after purification by Magne His?protein purification kit.Large,higher purity.2.Construction of PEDV S_493-708 and Omp16 double gene lactic acid bacteria expression vectorsIn this study,the synthetic plasmid p UC57-Omp16-S_493-708 was double-digested to obtain the target gene Omp16-S_493-708,which was inserted into the lactic acid bacteria secretion expression vector pVE5523 and transformed into Top10 to obtain the pVE5523-Omp16-S_493-708 plasmid.PCR After correct identification with enzyme digestion,pVE5523-Omp16-S_493-708 plasmid was electrotransformed into Lactobacillus ATCC393.The PCR and enzyme digestion identification results showed that the Lactobacillus pVE5523-with the Omp16-S_493-708 gene inserted was successfully constructed.Omp16-S_493-708/ATCC393.3.Oral immunogenicity of PEDV S_493-708 and Omp16 double-gene recombinant lactic acid bacteria vaccineFifty 6-week-old female C57/B6 mice were randomly divided into 5 groups,and the recombinant recombinant bacteria pVE5523-Omp16-S_493-708/ATCC393 and blank bacteria ATCC393 were administered at a dose of 0.2 m L/head.Inject Omp16-S_493-708protein+CFA/IFA,Omp16-S_493-708 protein,S_493-708 protein,strengthen the immunization once two weeks,and perform orbital blood collection for each group of mice 2 weeks after the second immunization,and determine the serum Samples of IgG subclasses（IgG1,IgG2a,IgG2b）antibody levels,serum IFN-γ,serum IL-4,serum IL-10.The serum IgG subclass assay results showed that:pVE5523-Omp16-S_493-708/ATCC393,Omp16-S_493-708 protein+CFA/IFA,Omp16-S_493-708 protein,and S493-708 protein were all capable of 2 weeks after the second immunization Induces the body to produce certain levels of specific serum antibodies against PEDV,and the body’s IgG antibody subclasses are mainly IgG1,followed by IgG2a and IgG2b;the cytokine assay results in the serum showed that:pVE5523-Omp16-S_493-708/ATCC393 The levels ofγ-interferon,IL-4 and IL-10 secretion in the serum of mice immunized with Omp16-S_493-708 protein+CFA/IFA group and Omp16-S493-708 protein group were significantly different from those of S_493-708 protein group.Significantly（P<0.05）,the pVE5523-Omp16-S_493-708/ATCC393 group,the Omp16-S_493-708 protein+CFA/IFA group,and the Omp16-S493-708 protein group were not significantly different from each other（P>0.05）.