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Construction and Application of the Integrated Gene Containing Multi-Mimotopes and VP2 of Infectious Bursal Disease Virus

Author: ZhouYu
Tutor: WangYongShan;FanHongJie
School: Nanjing Agricultural College
Course: Preventive Veterinary Medicine
Keywords: Infectious bursal disease Vp2 gene Multi-mimotopes Integrated gene Indirect ELISA
CLC: S852.65
Type: Master's thesis
Year: 2011
Downloads: 3
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Infectious bursal disease(IBD) is an acute, highly exposed, viral infectious disease which is caused by infectious bursal disease virus (IBDV) in chickens.Infectious bursal disease virus (IBDV) not only leads animals death, but also causes severe immunodepression of B cell response in chickens 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.Expl:Construction of the integrated gene containing multi-mimotopes and vp2 of infectious bursal disease virus,and its expression in insect cellsUsing gene splicing by overlap extension PCR (SOE PCR), a special integrated gene vp2-5e was acquired with a multi-mimotopes (5e) and vp2 of infectious bursal disease virus (IBDV), and then cloned into pFastBacHT donor plasmid. The recombinant donor plasmid pFastBacHT-vp2-5e containing the multi-mimotopes and VP2 was constructed and transformed into competent E.coli DH10Bac cells growing on LB plate which containing three kinds of antibiotics. After screening, the recombinant expression Bacmid pBacHT-vp2-5e was obtained and used to transfect insect cell Sf9 with Lipofectamine reagent to produce recombinant baculovirus vBac-vp2-5e. The Sf9 cells infected with vBac-vp2-5e could generate specific fluorescent light in the indirect immunofluorescence assay (IFA), the self-assembly virus-like particles (VLP) could be observed by electron microscopy, and the protein band of approximate 59 kD was detected in the western blotting.ExpⅡ:Application of the recombinant protein of the integrated gene containing multi-mimotopes and vp2 of infectious bursal disease virusTwo groups consist of SPF chickens were immunized by the integrated gene recombinant VP2-5E protein and the recombinant VP2 protein, respectively. At 14 days post primary immunization, the ELISA antibody titers of chickens immunized by the integrated gene recombinant VP2-5E protein was 1.746×103, providing 40% mortality from challenge with the virulent IBDV while those immunized by the recombinant VP2 protein was 50% because of 1.359×103 ELISA antibody titers. After 14 days after the booster vaccination, the mortality of those immunized by the integrated gene recombinant VP2-5E protein was 0%, and the recombinant VP2 protein was 10%. However, The ELISA antibody titers of chickens immunized by the integrated gene recombinant VP2-5E protein was up to 4.598×103 significantly higher than 4.044×103 of those immunized by the recombinant VP2 protein. The results suggested that the SPF chickens immunized by the integrated gene recombinant VP2-5E protein could resist the challenge with the virulent IBDV and be better than those immunized by the recombinant VP2 protein. An indirect ELISA for the detection of antibody against IBDV was established with the integrated gene recombinant VP2-5E protein. The recombinant VP2-5E protein was coated at 5μg/mL and serum samples were diluted at 1:400. The result of specificity test revealed VP2-5E antigen have no cross-reaction with positive sera of newcastle disease virus (NDV), infectious bronchitis virus (IBV), egg drop syndrome virus (EDSV), H9N2 influenza virus. Duplicate test showed the variation coefficients of the OD value of intra-assay and inter-assay to the assy were within 10%. About 200 serum samples collected from chiken farms were detected by the indirect ELISA and the commercial ELISA kit produced by IDEXX, respectively. The positive coincidence was 92.9%, the negative coincidence was 100%.The results indicated that the developed indirect ELISA had very high specificity and repeatability.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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