Prokaryotic Expression Of The Major Core Protein VP7 Of Bluetongue Virus And Preparation Of Monoclonal Antibodies

Bluetongue disease (BT) is an arthropod-borne viral disease affecting ruminants, caused by Bluetongue virus (BTV). At present, the fatality rate of ruminants affected BTV is about 30%,the rate of sheep reach up to 80%,and resulted in a serious economic loss. Bluetongue disease is a World Organization for Animal Health reportable (formerly list A)disease, and it is a Ministry of Agriculture of the Peoples Republic of China listⅠdisease. BTV is a double-stranded virus with a genome consisting of 10 dsDNA segments which encode seven structural proteins(VP1～VP7) and four nonstructural proteins(NS1,NS2,NS3 and NS3a).Four of all the seven structural proteins constitute a double layer capsid.The outer capsid is made of VP2 and VP5,and the inner capsid is made of VP3 and VP7.VP7 is conservative and there are group-specific antigenic sites mapped on it. And VP7 is the major core antigen of bluetongue virus, which can induce the production of group-specific antibodies against the virus. In this study VP7 of BTV was expressed in E.coli by a recombinant vector, and highly expressed VP7 for serological tests of BTV as a diagnostic reagent antigen. Two monoclonal antibodies (McAb) against VP7 of BTV were prepared.This study gives us a clue to research the diagnosis reagent.1. Expression and antigenetic characterization of VP7According to the published VP7 gene sequence of BTV on GenBank,A pair of primers were designed and synthesized. The VP7 gene of BTV-1 was amplified by RT-PCR, then was cloned into prokaryotic expression vector pMAL-c2X and sequenced. The length of VP7 gene was 1 050 bp, which encoded 349 amino acids. The recombinant plasmid pMAL-VP7 was obtained. The fusion protein (MBP-VP7) was produced after induced with 0.5 mmol/L IPTG from E. coli TB1 that was transformed with pMAL-VP7. The fusion protein is soluble and about 90 ku. The indirect enzyme-linked immunosorbent assay (ELISA) indicated that the expressed BTV VP7 was highly immunogenic.2. Preparation and identification of McAbs against VP7BTV1 was cultured in BHK-21 cell. The obtained virus was inactivated with 0.1% formaldehyde. Six months old Balb/c mice were immunized with the purified BTV antigens.Splenocytes from the immunized mice were fused with SP2/0 myeloma cells, and positive hybridoma clones were screened by ELISA. And limited dilution method was performed to subclone the positive clones. Two positive hybridoma cell strains designated 2F2C8 and 7D5H4 were obtained. The ELISA titer of culture supernatant were 1×105 and 1×104,and ascites were 1×108 and 1×107,respectively.These McAbs had good specificity to BTV1, and no cross-reaction was found when E.coli cell and BHK-21 cell were tested. McAb 2F2C8 purified from ascites had high activity of binding the recombinant fusion protein VP7,deteced by competitive ELISA with different serotypes of sheep anti-BTV antibodies.