Baculovirus Surface Display Of VP2 Envelope Protein Of Infectious Bursal Disease Virus And Its Immunogenicity

Infectious bursal disease (IBD) is an acute, highly exposed, viral infectious disease which is caused by infectious bursal disease virus (IBDV) in chickens. In recent years, IBDV mutant enhanced virulence and the emergence of a number of very virulent strain, the normal vaccine has failed to control the spread of this disease. In this case, the need to develop a more effective and more safe new vaccines against the disease becomes a focus for researchers.VP2 is the major structural protein of IBDV and the main host protective immunogen .It contains epitopes which can induce specific neutralizing antibody. Baculovirus surface display system have been developed rapidly in recent years.It is a new eukaryotic display system, and modificate the external source of protein such as glycosylation and other post-translational processing and folding. In view of these theoretical and technical basis and practical needs, we use the baculovirus insect cell expression system to display the VP2 protein of IBDV on the baculovirus membrane capsule. We obtained the following results:Design primer, VP2 gene was amplified using PCR.the gene was connected to the T vector by restriction enzyme digestion, recoveried the correct gene identified by agarose gel electrophoresis and sequencing and re-connect it to pFastbac SC transfer vector, constructed recombinant transfer vector BacSC-VP2 successfully. Transferred the vector into E. coli DH5αcompetent bacteria. After identified by agarose gel electrophoresis, PCR and restriction analysis,the correct BacSC-VP2 recombinant plasmid transformed into E. coli DH10 bac competent bacteria, recombinant plasmid shuttle vector Bacmid was successfully structured.Blue-white was used to screen recombinant bacmid, and sf9 insect cell was transfected with the recombinant bacmid. the cells showed green fluorescence in transfected sf9 insect cells with fluorescence microscopy;western-blot, immune electron microscopy and confocal laser microscopy were used to detect the expression of VP2 protein., the protein occurring at about 50 Ku band in western blot detection, consistent with the expected results; immune electron microscope, a colloidal gold particles deposited on the baculovirus surface, showed the expression of VP2 protein in the baculovirus membrane capsule. Laser scanning confocal microscope could detect VP2 protein in the cell membrane of sf9. Balb / c mice and chickens were immuned with recombinant baculovirus purified with high-speed centrifugation to detect the recombinant baculovirus. The results showed that in mice immunized with recombinant baculovirus: lymphocyte proliferation test showed: lymphocyte stimulation index SI was 1.526 witch indicate baculovirus significantly stimulated lymphocyte growth; to do with serum indirect ELISA, the test group antibody titer was 5000; serum neutralization test, the test group rat serum has significant neutralizing activity on the virus,neutralization titer was 1:32. Chicken serum ELISA : the recombinant baculovirus of chicken immune serum S / P values also showed positive; chicken bursals of no immunized but challenged group were significantly mucosal bleeding, swelling and lesions, and chicken bursal immunized was not obvious; above all, our preparation of recombinant subunit vaccine can induce specific antibodies, and has a protective effect on the body.