Cloning And Prokaryotic Expression Of BVDV Autoprotease P20 And Core Protein P14 Gene

Bovine viral diarrhea-mucosal disease virus (BVDV) represented the prototype member of the pestivirus genus, family Flaviviridae, mainly caused bovine viral diarrhea-mucosal disease. Bovine infected by BVDV showed diarrhea, acute and chronic mucosal diaease, persistent infection and immunotolerance, immunosupression, pregnant cow abortion, dead fetus and abnormal fetus. BVDV was a worldwide pathogen in cattle which had not been controlled by classical vaccination,and had seriously endangered the cattle herds.BVDV P20 and P14 gene located in the 5' end of the genome, encoded nonstructural protein of autoprotease and structural protein of core protein respectively. Core protein had antigenicity, while the antigenicity of autoprotease protein had not been reported. In order to meet the need of the application of genetically engineered subunit vaccine, BVDV P20 and P14 gene were cloned and expressed in prokaryotic expression system, whose aim was to lay a foundation for genetically engineered diagnostic antigen and to make a necessary preparation for elucidating the antigenicity of protein P20.Two sets of primers were disigned, according to the genomic sequence of BVDV Oregon C24V(AF091605) strain published in GenBank, for amplifying P20 gene of BVDV Osloss-Like strain in vitro by the method of RT-nested PCR. The product of PCR of P20 gene was cloned into pMD18-T vector for sequencing. Homologous analysis showed that the homology of nucleotide sequence of P20 gene between Osloss-Like strain and other strains, such as VEDEVAC(AJ585412), Osloss(M96687), Oregon C24V(AF091605), SD1(M96751), and NADL(AJ133738) strain was 99.21%, 93.65%, 80.95%, 79.96% and 78.97% respectively. On the base of the result of homology analysis of P20 gene, another set of primer was designed according to the gneomic sequence of BVDV Osloss strain published in GenBank, P14 gene was amplified specially with this set of primer. Sequencing and homologous analysis showed that the homology of nucleotide sequence of P14 gene between Osloss-Like strain and other strains, such as VEDEVAC(AJ585412), Osloss(M96687), SD1(M96751), Oregon C24V(AF091605), NADL(AJ133738) strain was 98.37%, 94.77%, 82.53%, 79.41% and 78.76% respectively. Phylogenetic analysis showed that the biogenesis of P20 and P14 gene between BVDV Osloss-Like strain and VEDEVAC strain was closer than between Osloss-Like and other strains.By recombinant DNA techniques, both P20 and P14 gene of BVDV Osloss-Like strain were subcloned into prokaryotic expression vector pGEX-6P-1 and pPROEX-HTb respectively to construct recombinant expression vector of pGEX-6P-P20, pPROEX-HTb-P20, pGEX-6P-P14 and pPROEX-HTb-P14. Then the recombinant expression plasmid above all were transformed into engineering bacteria of E.coli respectively, recombinant expression plasmid of pGEX-6P-P20 and pPROEX-HTb-P20 highly expressed after induced with IPTG, while the expression amount of recombinant expression plasmid of pGEX-6P-P14 was very low after induced with IPTG, and the expression of pPROEX-HTb-P14 was hardly detected. In order to achieve the expression of P14 gene in E.coli, differrnt expression conditions were performed on recombinant expression plasmid of pGEX-6P-P14 and pPROEX-HTb-P14. It was assumed that the protein expressed by P14 gene of BVDV Osloss-Like strain may be a kind of toxic protein and was toxic to E.coli of BL21(DE3)pLysS and DH5α.Postgraduate: Li HuixinSpecialty: Preventive Veterinary ScienceSupervisor: Prof.Wang Junwei...