Study On DNA Vaccine Of Pseudorabies Virus Glycoprotein D And Immune Enhancement Effect Of VP22 Protein On The Vaccine

Pseudorabies, also designated as Aujeszky's disease (AD), is a serious illness of swine and causes significant financial losses in the pig industry. To control AD and to reduce economic losses in the pig industry, active immunization with modified live or inactivated vaccines has been performed widely. Although these vaccines can provide protection against clinical signs of AD in swine to some degrees, they have certain defects, especially limited protective efficacy and the danger of reversion to a more virulent strain. Therefore, it is urgent to develop safer and more effective vaccines to control this disease. As a new immunology theory and method, Genetic immunization and nucleic acid vaccine or genetic vaccine developed basing on research of gene therapy, have gotextensive attention and great progress in recent years. As its advantages of safety, effectiveness, easily construction, etc. genetic vaccine has become the preferential potential method during research the prophylactic method against serious diseases.Based on clone, expression of Pseudorabies Virus(PrV)(Ea strain) major immunogenicity gD gene, we constracted the eukaryotic expression plasmida of it as DNA vaccine, detected the immune response level and protective efficacy of vaccine injected by muscle. The results demonstrated that DNA vaccine can stimulate the humoral and cellar immune response effectively, also indicated that immunopotentiation of VP22 to Pseudorabies DNA vaccine, the major research was the follow:1. Cloning of gD gene of PrV(Ea strain) and VP22 geneUsing recombinant plasmid pcDD and pMD-VP22 as template, designed primer which contain Hindlll and Pst I sites in the upstream and downstream of gD gene respectively, and lead-in Xbal and EcoR I sites of VP22 gene by polymerase chain reaction(PCR) amplification. Cloned the amplified gD and VP22 gene on the vector pUC119 to construct recombinant plasmid pUC-gD> pUC-VP22 and pUC-gD-VP22. The series of pUC-gD and pUC-VP22 were sequenced by Boya Biotechnology Company. The fragment of gD, VP22 and gD-VP22 gene were 1200bp , 760bp and 1200bp by digest electrophoresis. Sequencing result showed that the amplified fragment of gD, VP22 and gD-VP22 gene were 1266bp ,756bp and 2028. And found that two amplified gene fragment consistent with gD gene (AF086702 ) and VP22 gene (AY190527) of publicated in PrV, without base mutation.2. Construction of expression plasmidDigest pUC-gD, pUC-gD-VP22 and prokaryotic expression vector pET28a(+)and eukaryotic expression vector p3XFLAG-CMV^?-10 by restriction enzyme HindIII and EcoR I, link and construct prokaryotic expression plasmid pET-gD, pET-gD-VP22 and eukaryotic expression plasmid p3XFLAG-gD, p3XFLAG-gD-VP22. Analysis prokaryotic expression plasmid and eukaryotic expression plasmid through electrophoresis appeared two obvious straps on 1300bp and 2000bp, indicated the purpose gene were inserted into multiple clone site of expression vector.3. Expression of gD in E.coli.The prokaryotic expression vector pET-gD and pET-gD-VP2 transformed BL21(DE3), induced by IPTG, and the result of SDS-PAGE showed that there was a obvious specific strap at 47kDa in...