The Optimizing Expression Of E₂Gene Of BVDV In BCG And The Immune Efficacy Of Its Expressed Products

Bovine viral diarrhea-mucosal disease virus （BVDV） is the member of the pestivirus genus,family Flaviviridae, caused many clinical symptoms and disease. Bovine infected by BVDV showed diarrhea, acute and chronic mucosal diaease, persistent infection and immunotolerance, immunosupression, pregnant cow abortion, dead fetus, abnormal fetus, Respiratory syndrome, Congenital defects, Enteritis, Persistent infection （PI） and mucosal disease （MD）. Bovine viral diarrhea-mucosal disease was a worldwide pathogen with highly infection,and it has tendency to expand.There is no effective measure to control this disease now, and the best way to prevent beforehand is to study and research a kind of vaccine which is safe and available. BVDV E2is the best immunogenicity proteinum in structural proteins. It can induce neutralizing antibody. Also it adapt to the environment perfectly because of E2gene has high aberration rate. So E2gene is the first choice protective antigenic gene to the genetically engineering vaccine.The researchs showed that the BCG is utility preparation to Tuberculosis. BCG is not only a adjuvant but also a antigen. A single vaccination, availability of strong and long-lasting cellular and humoral immunity.On these grounds,this study adopt shuttle plasmid vector pMV361to construct rBCG expressing E2gene, researching the immunogenicity of protein expressed and the regularity of BVDV and BCG antibody in mouce,which is to obtain effective genetic engineering live vector vaccine of BVDV.The research amplified E2gene from BVDV Changchun184strain by PCR, then insert E2gene into pMD18-T plasmid and transfected it to competence of E.coli JM109and choosed positive clones. Sequencing result s showed that E2gene of changchun184strain was composed of1122bp nucleotides encoding374amino acid residues. The sequence was analyzed and t he epitopes of E2protein were predicted by bioinformatics sof twares. Epitopes coding region （1～297、1～345、1～374、45～297、45～345、45～374）of E2gene were amplified by PCR and cloned into pMD18-T and choosed positive clones. We extract recombination plasmid which can be identificated by electrophoresis,PCR, enzyme incide and sequence determine. Construct the shuttle plasmid vector pMV361-E2-1、 pMV361-E2-2、 pMV361-E2-3、 pMV361-E2-4、 pMV361-E2-5、pMV361-E2-6by connection of pMD18-T-E2recombinate plasmid and E.coli-Mycobacteria shuttle expression plasmid vector pMV261which had been enzyme incide by EcoR Ⅰ and Cla Ⅰ.After groped the main factors which can effect the result of electroporation,the best electroporation parameters were confirmed that Voltage:1.8kv、 Interval:800ms, Pulse:150μs. In view of the above, the resulting plasmids pMV361-E2was electrotransfected into BCG and selected by kanamycin. The results showed that the vector pMV361-E2₁、pMV361-E2-2、 pMV361-E2-3、pMV361-E2-4、pMV361-E2-5pMV361-E2-6containing each region of the E2gene were successful constructed, and Westernblotting indicated that t he recombinant proteins could react with polyclonal antibody against BVDV. the E2gene was successfully expressed in rBCG.Because of different region of the E2gene having different expressing ability of target gene,In order to obtain rBCG which express level of E2is higher, the research use this six kinds of rBCG vaccinated mouse separately.By means of ELISA to detect the specific antibody of BVDV and BCG in mouse,the results indicated that the tendency of BVDV and BCG antibody is in substantial agreement after immuning this six kinds of rBCG and causing the humoral immune respones and cellullar immunologic response,which showed that the pMV361-E2-1rBCG is better than Other groups.