Molecular Design Of The Chimera Of Ibdv Mimotope-Hbc And Its Immunologic Function

Infectious bursal disease virus (IBDV), the etiological agent of infectious disease, causes severe immunodepression of B cell response in chickens, mainly at the ages of3-6weeks, by destruction of lymphocytes in the bursa of Fabricius. Protection of chickens against IBDV is achieved by vaccinating breeding hens with conventional attenuated or inactivated IBDV vaccines. Because of the virulence change and antigenic epitope shift of the vvIBDV, a very virulent strain of the IBDV (vvIBDV) appeared and spread in our country causing sever economic losses. The occurrence of variants has been speculated to be due to the selection pressure from the live IBD vaccines administered.Young chickens vaccinated with the intermediate live attenuated IBD vaccines, which can induce moderate bursal atrophy, may have immunosuppression, interfering with vaccination against other diseases,also were hard to produce. With so mang disadvantages accociated with the currently available live attenuated vaccines against IBDV infection,the search for a new approach to improve the vaccine or to produce new vaccines is warranted. VP2is the main host-protective antigen of IBDV and carries major neutralizing eptitopes.Expl:Molecular Design and Immunogenic Analysis for the Chimera of IBDV mimotope and HBcAgTo enhance the immune efficacy of multi-mimotopes gene5epis of infectious bursal disease virus (IBDV), expression vector pET28-mHBc based modified-HBcAg with BamH1and EcoR1restriction sites was designed for developing chimera gene and constructed by means of overlap extension PCR technique. The multi-mimotopes gene5epis was inserted into mHBc of pET28-mHBc, then chimera expression plasmid pET-mHBc-5epis was achieved and transformed into E.coli BL21(DE3). After induction with IPTG, the recombinant chimera mHBc-5epis protein was efficiently expressed in E.coli. The result of SDS-PAGE showed that the molecular weight of the expressed chimera was approximately 29ku. The recombinant chimera protein reacted with anti-IBDV polyclonal antibody in western-blotting. In the supersonicated bacterial lysate, self-assembly virus-like particles (VLP) with diameter of100nm-120nm was observed by electron microscopy, and the titer reached to1:200with sandwich ELISA for the detection of IBDV antigen. This study demonstrated that the recombinant mimotope-based chimera protein promises to be a novel subunit vaccine strategy for IBD.ExpII:The immunologic function of the recombinant chimera mHBc-5epis protein in chickenThe chimera expression plasmid pET-mHBc-5epis was transformed into E.coli BL21(DE3) and induced with IPTG..The recombinant chimera mHBc-5epis protein was purified with SDS-PAGE, which was immunized to1-month SPF chicken. The contents of Th1type (IFN-γ, IL-2) and Th2type (IL-4, IL-6) cytokines in the serum were detected after the previous immunization by enzyme-linked immunosorbant assays (ELISA). On the second day, the contents of IL-2and IL-4were markly increased. The titer reached to1:200by indirect ELISA for the detection of IBDV antibody on the14th day after immunization. The results showed that HBcAg as the carrier can enhance the specific cellular and humoral immune responses in chicken. This is the first report that HBcAg can enhance the immunological protection of antigen epitope in chicken. The study supplies a foundation for the development of particle multi-epitope vaccine.