Preparation And Immune Protection Of The Recombinant VP2Protein Of IBDV

Infectious bursal disease (IBD) is one of the major infectious disease, which may cause serious economic loss both in our country and throughout the world. Now, there were security and manufacture defects in common ambly-toxicum vaccine and inactive vaccines, and much biosafety problems existing in the middle hadro-virulent live vaccines when applyed to protecting variational strain. So in practice, it is badly needed to develop a neotype IBD genetically engineering vaccine with better immune validity and biosafety, and even directly to the current popular strain. Two parts were content in this thesis:Expl. Preparation of the recombinant Vp2protein of infectious bursal disease virusThe efficiently expressed IBDV Vp2restructuring baculovirus vBacA-Vp2(P3generation) constructed in our laboratory in the early days were used to vaccinate the Sf9cells. After that the Sf9cells infected with rBacA-Vp2were stained positively against IBDV antibody using the indirect immunofluorescence assay (IFA). The IBDV antibody sandwich ELISA results showed positive reaction. Western blotting showed that the calculated protein of approximately53ku was in the expressed in the insect cells. Moreover, virus-like particles (VLPs) structure in the infected Sf9cells was observed under electron microscopy. The recombinant Vp2protein was self-assemblyed into virus-like particals. Our experiment provided the backgrounds of neotype virus-like partical vaccine and detective reagent researching directly at the recent prevalent IBDV. The Vp2protein expression condition optimized and Sf9cells pre-layed one day before intfection, vBacA-Vp2with multiple infections was vaccinated until the cell density reaching90%. Then cell culture was collected at72,96and120h respectly, the IBDV double-antibody sandwich ELISA technique was used to determine the antibody titer of the culture. Results indicated that the multiply of infection immunized with vBacA-Vp2was0.01, at the expression time point of 72h, and the recombinant Vp2protein content in the cell culture was the highest with the antigen titer upto3.2×103. Then the optimized culture condition of the recombinant Vp2pretein was used to vaccinate vBacA-Vp2, collecting the cell cultures, a great mass of the recombinant Vp2protein was produced, and to prepare the antigen for new and high efficient IBD virus-like particles genetical engineering vaccine.Exp II. Immune protection of the recombinant Vp2protein of infectious bursal disease virusThe lysate of Sf9insect cell infected by the recombinant baculovirus vBacA-Vp2was emulsificated with ISA206, ISA50and whte oil respectly, made into antigen and used to immunize2-week old SPF chicken,14days post primary immunization, the level of enzyme linked immunosorbent assay (ELISA) antibody titers of chicken immunized by the recombinant IBDV Vp2protein plused with ISA206and B87vaccine groups were significantly higher than others (ISA50, white oil and blank control groups)(P<0.01). However, no obvious difference was found between ISA206and B87groups (P>0.05).14days post the second immunization, the level of enzyme linked immunosorbent assay (ELISA) antibody in the group immunized by the recombinant Vp2protein plus ISA206immunologic adjuvant was significantly higher than other3groups (B87, ISA50and blank control groups)(P<0.01), while no obvious difference was observered among the chicken immuned with the traditional B87vaccine and the recombinant Vp2protein plus white oil and ISA50(P>0.05). Compared with other groups (B87, ISA50, white oil and blank control groups), the results of neutralizing antibody titer assay showed that the neutralizing antibody titer in ISA206group significantly increased (P<0.01). After challenging, chicken in ISA206, white oil and B87vaccine groups were all alived with a survival rate of100%, while the rate was80%in chicken protected by the recombinant Vp2protein plus ISA50immunologic adjuvant, and the ckicken in the blank control group were all died. During the7days observation, no clinical symptom and pathological change were found in the ckicken protected by the recombinant Vp2protein plus ISA206and white oil immunization. It was concluded that the IBD virus-liked particles subunit vaccine prepared by the recombinant Vp2protein showed an important application prospect on the researching in new type IBD genetical engineering vaccine.