Expression Of The Major Core Protein VP7 Of Bluetongue Virus In E.coli And Preparation Of Monoclonal Antibodies

Bluetongue disease (BT) is an arthropod-borne viral disease affecting ruminants,caused by Bluetongue virus (BTV). At present, bluetongue disease is listed in A infectiousdiseases by World Organization for Animal Health (OIE). The disease broke out primarilyin tropical and temperate regions of the world, and resulted in a serious economic loss.VP7 is the major core antigen of bluetongue virus, which can induce the production ofgroup-specific antibodies against the virus. And there are group-specific antigenic sitesmapped on the VP7. In this study group-specific antigen VP7 of BTV was expressed inE.coli by a recombinant vector, and highly expressed recombinant VP7 for serologicaltests of BTV as a diagnostic reagent antigen. Two McAbs against group-specific antigenVP7 of BTV were prepared. This study gives us a clue to research the diagnosis reagent.1. Expression and antigentic characterization of the major core protein VP7 ofBluetongue virusAccording to published VP7 gene sequence of BTV, primers were designed andsynthesized. The complete cDNA clone of VP7 gene in the cloning procedure wasamplified by RT-PCR. The group-specific antigen VP7 gene of BTV was cloned intopGEM-T Easy vector and sequenced. The length of VP7 gene was 1050 bp, whichencoded 349 amino acids. The recombinant vector containing BTV VP7 cDNA wasconstructed. Then the fragment of VP7 gene was subcloned into prokaryotic expressionvector pET-DsbA,pET-GST and pET-His by PCR and restriction endonucleases. Therecombinant expression vectors were transformed into E.coli BL21 (DE3) pLysS andinduced by IPTG. The higher expression vector pET-DsbA/VP7 was screened. TheHis-tagged recombinant protein was purified using a His-tag affinity chromatographycolumn on Ni²⁺-nitrilotriacetate (NTA) resin. The results of SDS-polyarcylamide gelelectrophoresis (SDS-PAGE) and Western blotting revealed that the recombinant fusionprotein was expressed with a molecular mass of approximately 63.2 kDa. The indirectenzyme-linked immunosorbent assay (ELISA) indicated that the expressed BTV VP7 washighly immunogenic.2. Preparation of Monoclonal Antibodies against serogroup-specific antigen VP7 ofBluetongue virusBTV-5 was cultured in BHK-21 cell. The obtained virus was inactivated with 0.1% formaldehyde. Balb/c mice were immunized with the purified BTV antigens. Splenocytesfrom the immunized mice were fused with SP2/0 myeloma cells, and positive hybridomaclones were screened by ELISA and limited dilution method was performed to subclonethe positive clones. After three cycles of subcloning, two McAbs against the VP7 ofBTV-5 were obtained, designated as 3E2 and 1Cll. The ELISA titer of culturesupernatant were 2⁹ and 2¹⁰, and ascites were 4×10⁶ and 16×10⁶, respectively. TheseMcAbs had good specificity to BTV-5. No cross-reactions were found when epizootichemorrhagic disease virus (EHDV), BHK-21 cell, and E.coli cell were tested. McAb 3E2purified from wild waterfowl had high activity of binding the recombinant fusion proteinVP7, detected by competitive ELISA with different serotypes of sheep anti-BTVantibodies.