Expression Of Pp38 Gene Of Marek's Disease Virus RB1B Strain And Development Of Monoclonal Antibodies To Pp38 Protein

pp38 gene of Marek's Diseas Virus (MDV) was amplified by polymerase chain reaction (PCR) . The products of PCR were cloned into pGEM-T easy vector. Positive recombinant clone named pp38-T was identified by restrict enzyme digestion and sequencing. The fragment of pp38 was subcloned into baclovirus tranfer vector pFastBac1. The transfer vector pFastBacl-pp38 was transformed into DH10Bac E. coli cells by site-specific transposition. PP38 gene was integrated into Bacmid and recombinant shuttle vector named Bacmid-pp38. The recombinant baculovirus, name as reBac-pp38, was obtained from Sf9 cells transfected with shuttle vector Bacmid-pp38 mediated by lipofectin. rpp38 was expressed in the Sf9 cells at 96h post-infection with reBac-pp38, which was identified by IFA and with polyclonal antibodies to MDV.A strain of monoclonal antibody (McAb) against MDV pp38 protein was obtained by cell fusion between SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with Sf9 cells infected with reBac-pp38. The hybridoma culture supernatant was screened by indirect immunofluorescent assay (IFA). McAb named as pp38-2F6 was developed. The McAb reacted with CEFs infected with MDV-RB1B or MDV-CVI988 in IFA but not with other viruses. The subtype of immunoglobulin of McAb is IgG2b for pp38-2F6 in capture ELISA. The results showed that McAb would be very useful for identification of the expression of MDV-pp38 protein and investigation of the function of PP38 protein.