Cloning And Prokaryotic Expression Of VP2 Gene Of Infectious Bursal Disease Virus Henan H-1 Strain

Infectious bursal disease(IBD)is a high tangent infectious disease caused by infectious bursal disease virus (IBDV). Pre-B-lymphoid cells or immature B-lymphoid cells, the target of IBDV, often appear degeneration and necrosis. Since the bursa of Fabricius is important organ to the maturation of B-lymphoid cells , a mount of B cells in the lymphoid follicle of bursa Fabricius is lytic , atrophy of bursa causing immunosuppression, leading to increased susceptibility to other pathogens and decreased immunological response to vaccine(new castle disease and avian influenza), producing considerable economic loss in poultry industries.VP2 protein is the main structure protein and protective antigen of IBDV, containing the B cell epitopes inducing the neutralization antibodies. The main contents are as follows: reverse transcription-polymerase chain reaction(RT-PCR)was used to amplify VP2 gene from identified IBDV field isolate H-1 isolated from a poultry farm in Henan. The amplified VP2 gene was cloned into prokaryotic ( Escherichia coli) expression system to produce VP2 proteins.In this study, RT-PCR was conducted after isolating total RNA of IBDV , and the 1461 bp VP2 gene was amplified .The VP2 gene was then cloned into the pGEM-T vector. Recombinant plasmid pTVP2 was constructed successfully. Sequence analysis showed that the high homology of target gene and animo acid with the vvIBDVs from Japan and Europe. The similar variation of characteristic amino acid implied strain H-1 was probably vvIBDV . Phylogenetic analysis showed H-1 was in the same lineage to vvIBDV UK661 and OKYM isolated from Europe and Japan respectively, implying H-1 might be vvIBDV.The VP2 gene in pTVP2 vector was subcloned into prokaryotic expression vector pET-28a. After double enzyme digestion and PCR, the vector pETVP2 was successfully constructed. The 53.7 Kd recombinant protein was expressed with high efficiency in E.coli after induction with IPTG.. The expressed protein in form of inclusion body showed specific immunoreactivity with chicken polyclonal antiserum to IBDV in western blot assays. The research above provided a basis for studying on the role of VP2 protein and establishing related diagnosis system .