Expression Of VP2 Gene Of Infectious Bursal Disease Virus And Preparation Of Monoclonal Antibodies Against The Recombinant VP2 Protein

Infectious bursal disease virus (IBDV), the etiological agent of infectious disease, destructs lymphatic tissue, especially the lymphocytes in the bursa of Fabricius in young chickens. It causes death and immunodepression, and results in immunity decrease and vaccination failure. This disease continues to pose significant losses in the commercial poultry industry. The epidemic situation of IBD is complex, while the vIBDV (virulent IBDV) and vvIBDV (very virulent IBDV) appeare for the past few years which can escape from common vaccinal immounization, because of the virulence and antigencity changing. IBDV VP2 protein is the main structural protein and host protective antigen, therefore, studies on VP2 protein and the detection methods for VP2 are important works at present. In this study, we have expressed the prevalent IBDV VP2 gene in Escherichia coli and baculovirus expression system respectively. Nine monoclonal antibodies (mAbs) against VP2 protein were developed. The thesis includes 3 parts:1. Expression in Escherichia coli of VP2 gene of infectious bursal disease virusA VP2 gene of infectious bursal disease virus (IBDV) was amplified from bursa of Fabricius of chickens with immunoprophylaxis defeat in Anhui province and cloned into the prokaryotic expression vector pGEX-4T-1. The expression plasmid pGEX-VP2 was constructed and transformed into competent Escherichia coli Rosetta (DE3), the target protein was expressed under the induction with IPTG and was positive in the detection of IBDV sandwich ELISA. SDS-PAGE analysis showed that the VP2 fusion protein was approximately 68 ku in molecular weight, and it made up 16.0% of the total bacterial proteins as inclusion bodies in E. coli. Western-blotting showed that the VP2 protein could react with IBDV antibody, indicating that the VP2 protein possessed good antigenicity. 2. Expression of VP2 gene of infectious bursal disease virus in insect cellsTo develop the recombinant VP2 protein of the current infectious bursal disease virus (IBDV), the VP2 gene of IBDV isolated from chicken with immunoprophylaxis defeat in Anhui province was cloned into pFastBacHTA donor plasmid. The recombinant donor plasmid pFastBacHT-VP2 containing VP2 was constructed and transformed into competent E. coli DH10Bac cells. After screening, the recombinant expression Bacmid rBacHT-VP2 was obtained and used to transfect insect Sf9 cells with Lipofectamine reagent to produce recombinant baculovirus vBacHT-VP2. The protein band of approximate 53 ku was detected in western blotting, the Sf9 cells infected with vBac-VP2 could generate specific fluorescent light in the indirect immunofluorescence assay (IFA), and the self-assembly virus-like particles (VLPs) could be observed by electron microscopy.3. Preparation and application of monoclonal antibodies against VP2 protein of infectious bursal disease virusRecombinant VP2 protein of infectious bursal disease virus (IBDV) of AH1 strain expressed by Escherichia coli and recombinant baculovirus and purified IBDV antigen were used to immunize BALB/c mice, nine hybridomas secreting anti-VP2 monoclonal antibodies(mAbs) were established by hybridoma technique. The antibodies titers were 4×10-2～1.6×10-3 in culture supernatants and 10-2～10-5 in ascetic fluids by indirect-ELISA. The recombinant VP2 protein could react with four mAbs and the specific protein bands were 53 ku in western-blotting. Additivity ELISA indicated that A12G,B12F and F9C were corresponding to the same antigenic epitope, and the other six mAbs were different. The nine mAbs were specific to IBDV and didn't react with NDV, IBV, EDAV, AIV H9N2, culture supernatant of Sf9 cells and lysate of CEF in sandwich ELISA. The stability experiment indicated that the nine mAbs posed the ability of secreting antibodies after subculturing, freezing and thawing test. The sandwich ELISA established by the mAbs for detecting IBDV showed highly specificity.