Eukaryotic Expresstion Of BVDV E～(rns) Gene And Construction Of Eukarytic Expression Vector Of E2 Gene

Bovine viral diarrhea-mucosal disease virus(BVDV)is the member of the pestivirus genus, family Flaviviridea, mainly caused bovine viral diarrhea-mucosal disease. Bovine infected by BVDV showed diarrhea, acute and chronic mucosal disease, persistent infection and immunotolerance, immunosupession, pregnant cow abortion, dead fetus and abnormal fetus. BVDV can infect catties, calf and other animals. BVD was a worldwide pathogen in cattle with highly infection ratio, it had not been controlled by classical vaccine, and had serious endangered the cattle herds. BVDV has infected in more than 20 provinces of China, and it has tendency to expand.BVDV E^rnsand E2 gene located in the 5 'end, one third of the genome, encoded structural protein of envelope glycoproteins E^rns and E2 which can induce neutralization antibody to resistent BVDV and CSFV infection. So it had important value to make diagnostic reagent using E^rns and E2.In this study, after digesting by BamH I and HindIII of recombinant plasmid pMD18-T-E2 that our lab owned, E2 gene was subcloned to the baculovirus transfer vector pBlueBacHis2A, such construct recombinant plasmid pBlueBacHis2A-E2. As the same, E2 gene was subcloned to P.Pastoris transfer vector pPIC9K, and recombinant plasmid pPIC9K-E2. was constructed. PCR product of BVDV E^rns gene that our lab owned was cloned to pMD18-T, a vector identification, E^rns gene was cloned to baculovirus transfer vector pBlueBacHis2A, then, recombinant plasmid pBlueBacHis2A-E^rns was constructed. Bac-N-Blue and pBlueBacHis2A-E^rns were co-transfected into insect cell Sf9. By plaque assay, the recombinant baculovirus are obtained. By analysis of SDS-PAGE and Western -blotting, the target protein can be identified and detected. T he MW of the recombinants protein was approximately 30kDa and presented strong antigenicity.E^rns gene was cut with restriction enzyme EcoR I and Not I from the recombination plasmid pBlueBacHis2A-E^rns, and subcloned into the expression vector pPIC9K, which was identified by enzyme digestion and PCR amplification. The recombination expression vector, pPIC9K-E^rns, was linearized by Sal 1 and electroporated into P.PastorisGS115. The recombinant plasmid GS115-pPIC9K- E^rns was obtained by selected.Expression of BVDV E^rns fusion protein was followed by recombinant GS115-pPIC9K-E^rns with the induction of 1% methanol at 30℃, shaking at 200r/min. A...