Preparation And Identification Of Monoclonal Antibodies Against E2 And NS3 Protein Of BVDV

BVD is a disease caused by BVDV. Bovine's symptom infected by BVDV is diarrhea, acute and chronic mucosal disease, persistent infection, immunosupression and immunotolerance, pregnant cows'abortion, abnormal fetus and dead fetus. BVDV can infect cattles, pigs, calves, goats, camel and many other wild animals. BVD is wild spread in the world. BVD had severe impact on the cattle industry ,because of reproductive system'pathological changes and persistent infection by BVDV. BVDV and classical swine fever virus, sheep border diarrhea virus and some unclassified virus belong to Flaviviridae. BVDV genome has a large open reading frame and ambilateral untranslated regions, coding ten or eleven species proteins.E2 is one of the structural proteins of BVDV, which has the best immunogenicity and unconservation. Near the N-terminal half ,E2 contains a conformational antigen areas. The N terminatio of E2 is out of BVDV's envelope, which is the main antigen areas of BVDV. Also it is associated with BVDV antibodys'binding, mediating neutralization reaction and host cell recognition, the main adsorption site. There are antigenic determinants to distinguish pestivirus on the protein. BVDV and CSFV are highly homologous with the similar infection spectrum, which has brought much difficulties to the differential diagnosis of the disease. Therefore, BALB/c mice were immuned by BVDV pcDNA3.1-E2 in this experiment. Three monoclonal antibodies named 2E2, 3D12, 4D6 were gained.with purified BVDV as a screening antigens and the four subclones. 2E2 and 4D6 strains are IgM, 3D12 is IgG2a, withκchain. Werstern blot and indirect immunofluorescence indicated: 3 monoclonal antibodys are against the E2's conformational epitopes, and2E2 and 4D6 were only against BVDV. The epitopes binding to 2E2 and 4D6 are not owned by both BVDV and CSFV. The three McAbs are the material basis of method for the establishment of differential diagnosis.NS3 is one of the BVDV non-structural proteins, is mainly responsible for viral poly-proteins post-translational processing to generate mature viral proteins, is a prerequisite for self-replicating virus. Its coding region is the most conservative in the pestivirus, which is an immune advantage protein.The antibodys against NS3 could be detected in the animals immuned vaccine or infected BVDV. Currently BVDV-NS3 monoclonal antibody was not reported in China. In this study, BALB/c mice were immunized by NS3 protein, and five monoclonal antibodies named 1C8, 2H4, 4B5, 4F6, 4F8 were gained, and the identification of antibody's type showed: 1C8, 4F8 were IgG2b; 2H4 were IgG1; 4B5, 4F6 were IgG2a, withκchain. Five monoclonal antibodys titers were higher, Western blot showed: five monoclonal antibodys can specifically bind to the recombinant NS3 ,which provided materials for the establishment of test method and the research of NS3 proteins.