Construction And Research Of TGEV Bicistronic DNA Vaccine Carrying S Gene And N Gene

Transmissible gastroenteritis of swine(TGE) is a important enteric infectious diseases caused by transmissible gastroenteritis virus(TGEV).It becomes the most important disease threat the pig industry.The N and S protein of TGEV can induce generating of specific antibody.To construct a coexpression vector have considerable value in biology.It is a valid and convenient method to construct a diplpromotor vector. Preparing polyad nucleic acid vaccine with diplpromotor vector have significance in research of genetic immunization.In this study,the diplpromotor vector pVAXD was constructed and been authenticated.The vector pVAXD-N-Sa was constructed by inserting Sa and N into pVAXD,then we researched its immunogenicity initially.1.Construction and verification of eukaryotic bicistronic expression vector pVAXD.The vectors pVAXA and pVAXB were constructed by reforming the MCS of pVAX1.The PCR product of-PCMV-MCS-BGHpolyA-from pVAXA was inserted into pVAXB to form diplpromotor vector pVAXD.The DsRed gene and EGFP gene were inserted into pVAXD to construct vectors pVAXD-DsRed,pVAXD-EGFP and pVAXD-EGFP-DsRed.The recombinants were transfected and transiently expressed in COS-7 cells and detected by fluorescence microscope.pVAXD-EGFP-DsRed could coexpress DsRed and EGFP,and the fluorescence intensity had no significant deviation.The fluorescence intensity was similar between monocistronic and bicistronic vectors.The results show that the bicistronic vector pVAXD is successfully constructed,and it can express two genes simultaneously,which lay foundation for the advance research of the bigem DNA vaccine.2.Construction and verification of bicistronic vector pVAXD-N-Sa.The Sa fragment and N gene of TGEV were inserted into pVAXD to construct vectors pVAXD-Sa,pVAXD-N and pVAXD-N-Sa.The recombinants were transfected and transiently expressed in COS-7 cells and detected by RT-PCR.The length of the product of group pVAXD-N was 1.2kb.The length of the product of group pVAXD-Sa was 2.1kb.The group of pVAXD-N-Sa had two products,and the length were 1.2kb and 2.1kb.The results indicate that the N and Sa in pVAXD-N-Sa have transcribed,and it has been comed true initially of coexpressing N and Sa in vitro.3.Immunogenicity of pVAXD-N-Sa.Immunized the Jimpy mice with the recombinants pVAXD-N,pVAXD-Sa,pVAXD-N-Sa and pVAXD-N+pVAXD-Sa.An indirect enzyme-linked immunosorbent assay(ELISA) to detect antibodies in mice was developed.The antibody of TGEV was determinded at the 14^th day in group pVAXD-Sa,pVAXD-N-Sa and pVAXD-N+pVAXD-Sa,and the antibody had a peak at the 35^th day.The antibody of TGEV was determinded at the 28th day in the group pVAXD-N,and the antibody had a peak at the 42^th day.The antibody of TGEV induced by pVAXD-N-Sa and pVAXD-N+pVAXD-Sa were higher than those induced by pVAXD-N and pVAXD-Sa,and the antibody induced by pVAXD-N-Sa and pVAXD-N+pVAXD-Sa had no conspicuous difference.The results indicate that the antigen genes in the recombinants have expressed in vivo and induced humoral immunoresponse of mice.Joint action of N and Sa was more effective than using N or Sa alone.All these works will establish a foundation for advanced development of bigem DNA vaccine of TGEV.