Expression Of VP2 Gene Of Infectious Bursal Disease Virus And Development Of Monoclonal Antibodies Specific To VP2 Protein

VP2 gene of infectious bursal disease virus (IBDV) was amplified by polymerase chain reaction (PCR) from plasmid VP2-T. The products of PCR were cloned into pGEM-T easy vector. Positive recombinant clone named VP2-T4 was identified by restriction enzyme digestion and sequencing. The fragment of VP2 was subcloned into baclovirus tranfer vector pFastBac1. The transfer vector pFastBacl-VP2 was transformed into DH10Bac E.coli cells by site-specific transposition. VP2 gene was integrated into Bacmid and recombinant shuttle vector named Bacmid-VP2. The recombinant baculovirus, designated as reBac-VP2, was obtained from Sf9 cells transfected with shuttle vector Bacmid-VP2 mediated by lipofectin. rVP2 was expressed in the Sf9 cells at 72h post-infection with reBac-VP2, which was identified by IFA and Western blot with anti-VP2 monoclonal antibody 5D1.A set of monoclonal antibodies (McAbs) against infectious bursal disease virus (IBDV) VP2 protein was obtained by cell fusion between SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with IBDV. The hybridoma culture supernatant was screened by indirect immunofluorescent assay (IFA). Three monoclonal antibodies named as 1B5, 2H11 and 5D1 were developed. All of these monoclonal antibodies reacted only with Sf9 cells infected with rVP2 and CEFs infected with IBDV, not with CEFs infected with MDV, REV and Sf9 cells expressing rBacmid-vIL8 in IFA. The subtype of immunoglobulin is IgG2b for 1B5 and IgG1 for 2H11 and 5D1 in capture ELISA. The ascites titer of the McAb was 1:64000 in IFA and 1:80000 in indirect ELISA. The results showed that these monoclonal antibodies were specific to VP2protein of IBDV, and they would be very useful for identification of the expression of IBDV VP2 protein and investigating of the function of VP2 protein.