Cloning And Prokaryotic Expression Of BVDV Envelope Proteins E0,E1 And E2 Gene

Bovine viral diarrhea-mucosal disease virus (BVDV) is the 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.So it is of great interest to study the pathogen from the molecular biology. BVDV E0,E1 and E2 gene located in the 5'end,one third of the genome, encoded structural protein of envelope glycoproteins E0,E1 and E2 respectively. E0 and E2 had antigenicity, while the antigenicity of E1 had not been reported. One set of primers were disigned, according to the genomic sequence of BVDV Osloss(M96687)strain published in GenBank, for amplifying E0 gene of BVDV VEDEVAC-Like strain in vitro by the method of RT-PCR. The product of PCR of E0 gene was cloned into pMD18-T vector for sequencing. Homologous analysis showed that the homology of nucleotide sequence of E0 gene between VEDEVAC-Like strain and other strains, such as VEDEVAC(AJ585412), Osloss(M96687), Oregon C24V(AF091605), SD1(M96751), and NADL(AJ133738) strain was 99.41%~80.91%. On the base of the result of homology analysis of E0 gene, tow sets of primers were designed according to the genomic sequence of BVDV VEDEVAC strain published in GenBank, E2 gene was amplified specially by the method of RT-nested PCR. Sequencing and homologous analysis showed that the homology of nucleotide sequence of E2 gene between VEDEVAC-Like strain and other strains, such as VEDEVAC(AJ585412), Osloss(M96687), SD1(M96751), Oregon C24V(AF091605), NADL(AJ133738) strain was 98.58%~72.82%. One set of primer were disigned, according to the result of sequencing of E2 gene of VEDEVAC-Like strain and the genomic sequence of BVDV VEDEVAC(AJ585412) strain published in GenBank, for amplifying E1 gene of BVDV VEDEVAC-Like strain in vitro by the method of RT-PCR. Homologous analysis showed that the homology of nucleotide sequence of E1 gene between VEDEVAC-Like strain and other strains, such as VEDEVAC(AJ585412), Osloss(M96687), Oregon C24V(AF091605), SD1(M96751), and NADL(AJ133738) strain was 100%~76.75%.Phylogenetic analysis showed that the biogenesis of E0,E1 and E2 gene between BVDV VEDEVAC-Like strain and VEDEVAC strain was closer than between VEDEVAC-Like and other strains. By recombinant DNA techniques, E0,E1 and E2 gene of BVDV VEDEVAC-Like strain were subcloned into prokaryotic expression vector pGEX-6P-1 respectively to construct recombinant expression vector of pGEX-6P-E0, pGEX-6P-E1 and pGEX-6P-E2. Then the recombinant expression plasmid above all were transformed into engineering bacteria of E.coli respectively, recombinant expression plasmid of pGEX-6P-E0, pGEX-6P-E1 and pGEX-6P-E2 successfully expressed after induced with IPTG. In the experimentation, BVDV E0,E1 and E2 gene were cloned and expressed in prokaryotic expression system, whose aim was to lay a foundation for genetically engineered subunit vaccine and genetically engineered diagnostic antigen and to make a necessary preparation for elucidating the antigenicity of protein E1.