Isolation And Identification Of BVDV/GS01/2010Strain And Construction Of GE-/TK-Recombinant PRV Expressing E2

Bovine viral diarrhea/mucosal disease (BVD/MD), an important disease of domestic cattle, belongs to the genus pestivirus of the family Flaviviridae. Bovine viral diarrhea virus (BVDV) is the causative agent of BVD, an Office International des Epizooties (OIE) list disease of cattles that leads to important economic losses worldwide. Complex clinical symptoms of the disease lead to the difficulty for its treatment and prevention and controlling. Vaccination is an effective means of controlling the BVD, but so far there is no self-created BVDV vaccine. Certainly, the development of an efficient, stable, secure, convenient new vaccine is an urgent problem for the prevention and controlling of the disease, and pseudorabies virus (PRV) as the carrier used for the development of new animal BVDV vaccine provides broad ideas. Therefore, the BVDV of Gansu popular strains BVDV/GSO1/2010was isolated and identified, and the structural protein E2of BVDV/GSOl/2010strains was analyzed. In addition, gE-/TK-recombinant PRV expressing EGFP and BVDV-E2was constructed. The results were as the following:1. Treated materials of blood from Gannan Tibetan Autonomous Prefecture in Gansu province were passaged in MDBK cells. As a result, a BVDV strain was isolated and named BVDV/GSO1/2010. And BVDV cases recurred after artificially inoculating health cows with BVDV/GSO1/2010. The homology of5’-UTR gene sequences between BVDV/GSO1/2010and other member of BVDV family was analysed by biology software. The results showed that:the isolation strain belongs to the cytopathic effect of BVDV-1biotypes, the conclusion was the same to the target animal cases.2. Structural protein E2gene of BVDV/GSO1/2010strain was amplified by RT-PCR, cloned and sequenced, E2gene were analysed by bioinformatics software, the analysis and prediction of E2gene including nucleotide homology, amino acids homology, protein hydrophilicity and epitope. The results show that:Structural protein E2of BVDV/GSO1/2010strain is rich in secondary structure, and high antigenic region, containing more potential epitopes, including2～11,13～24,31～43,58～66,79～103,144～181,224～249,302～316,321～325,332～342,369～375aa region. Compared with reference sequence of BVDV-lfrom GenBank data, the homology of the E2nucleotide sequences were between74.1%～88.0%, the homology of the deduced amino acid of E2amino acid sequences was76.9%～90.6%; Based on the phylogenetic analysis, BVDV/GSO1/2010has the closest relationship of genetic distance with1-NADL, Singer-Arg, Oregon C24V and Shihezi strain.3. Transfer vector of pTK-/EGFP was constructed using homology recombination arms of TK gene of PRV BarthaK61and EGFP, which were successfully amplified by PCR from PRV genome and pEGFP-N1, both of them were inserted pQCXIX vector. This might be contributed to developing PRV virulence-deleted genetically engineering vaccine for animal diseases.4. Transfer vector of pTK-/EGFP/E2was constructed using E2gene of BVDV/GSO1/2010, which were inserted pTK-/EGFP vector. Co-transfecting vector of pTK-/EGFP/E2and genome DNA of PRV BarthaK61into BHK21cells, recombinant PRV/gE-/TK-/EGFP/E2was obtained following plaque screening. This might be contributed to developing BVDV and PRV divalent genetically engineered vaccine for animal diseases.