IBDV模拟表位与vp2组合基因的分子构建及其在昆虫细胞中的表达与应用
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摘要
鸡传染性法氏囊病(infectious bursal disease, IBD)是由鸡传染性法氏囊病病毒(infectious bursal disease virus, IBDV)引起的鸡的一种急性、高度接触性传染病。该病不仅引起患病动物死亡,而且还导致机体免疫抑制,使机体的免疫防御能力降低和疫苗免疫接种失败。对易感鸡群实施疫苗接种是预防该病最有效的方法,但由于各地流行的IBDV毒株的毒力与抗原性的差异,给疫苗毒株的选择造成较大的困难,每年因IBD发病死亡、免疫抑制、免疫失败造成的经济损失巨大。目前广泛应用中等毒力的IBD活疫苗,给IBD的防控带来极大的隐患,灭活疫苗存在着抗原制备的困难。因此,进一步研制新型基因工程疫苗,仍是当前禽病防控工作中亟待解决的重要课题。
试验1：IBDV模拟表位与vp2组合基因的分子构建及其在昆虫细胞中的表达
运用PCR重叠延伸技术将传染性法氏囊病病毒(IBDV)多表位5e与vp2基因连接构建组合基因vp2-5e,将其插入到杆状病毒表达系统供体质粒pFastBacHT中,转化E.coli DH10Bac感受态细胞,经三抗性筛选获得重组杆状病毒表达质粒pBacHT-vp2-5e,用脂质体法转染Sf9细胞,获得重组杆状病毒vBacHT-vp2-5e。对vBacHT-vp2-5e感染的Sf9细胞,以间接免疫荧光试验(IFA)检测,具有特异性荧光；电镜检查,重组VP2-5E蛋白能够自组装成病毒样颗粒(VLP)；用免疫印迹分析(Western-blotting),在59kD处出现一条特异性蛋白条带。
试验2：IBDV模拟表位与vp2组合基因重组蛋白的应用
用IBDV模拟表位与vp2组合基因重组蛋白免疫SPF鸡,第一次免疫14d后,组合基因重组蛋白组的ELISA抗体效价为1746,攻毒死亡率为40%,而用单一VP2重组蛋白组的ELISA抗体效价为1359,攻毒死亡率为50%；第二次免疫14 d后,组合基因重组蛋白组ELISA抗体效价上升到4598,攻毒死亡率为0%,单一VP2重组蛋白组抗体效价为4044,攻毒死亡率为10%。实验结果表明,组合基因重组蛋白免疫鸡可以抵抗IBDV强毒攻击且优于单一VP2重组蛋白。以组合基因重组蛋白VP2-5E作为包被抗原,建立了IBDV抗体ELISA检测方法,重组抗原包被浓度为5μg/mL,被检血清样品1：400稀释。特异性试验表明,与新城疫病毒(NDV)、传染性支气管炎病毒(IBV)、减蛋综合征病毒(EDSV)、禽流感病毒H9N2亚型的阳性血清均无交叉反应；重复性试验结果表明,间接ELISA方法批内、批间变异系数小于10%。将本实验建立的方法与IDEXX公司ELISA检测试剂盒进行了比较,检测200份IBDV血清样品,阳性符合率为92.9%,阴性符合率100%。实验结果表明,本实验建立的IBDV抗体ELISA检测方法具有良好的特异性与重复性。
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.
试验1：IBDV模拟表位与vp2组合基因的分子构建及其在昆虫细胞中的表达
运用PCR重叠延伸技术将传染性法氏囊病病毒(IBDV)多表位5e与vp2基因连接构建组合基因vp2-5e,将其插入到杆状病毒表达系统供体质粒pFastBacHT中,转化E.coli DH10Bac感受态细胞,经三抗性筛选获得重组杆状病毒表达质粒pBacHT-vp2-5e,用脂质体法转染Sf9细胞,获得重组杆状病毒vBacHT-vp2-5e。对vBacHT-vp2-5e感染的Sf9细胞,以间接免疫荧光试验(IFA)检测,具有特异性荧光；电镜检查,重组VP2-5E蛋白能够自组装成病毒样颗粒(VLP)；用免疫印迹分析(Western-blotting),在59kD处出现一条特异性蛋白条带。
试验2：IBDV模拟表位与vp2组合基因重组蛋白的应用
用IBDV模拟表位与vp2组合基因重组蛋白免疫SPF鸡,第一次免疫14d后,组合基因重组蛋白组的ELISA抗体效价为1746,攻毒死亡率为40%,而用单一VP2重组蛋白组的ELISA抗体效价为1359,攻毒死亡率为50%；第二次免疫14 d后,组合基因重组蛋白组ELISA抗体效价上升到4598,攻毒死亡率为0%,单一VP2重组蛋白组抗体效价为4044,攻毒死亡率为10%。实验结果表明,组合基因重组蛋白免疫鸡可以抵抗IBDV强毒攻击且优于单一VP2重组蛋白。以组合基因重组蛋白VP2-5E作为包被抗原,建立了IBDV抗体ELISA检测方法,重组抗原包被浓度为5μg/mL,被检血清样品1：400稀释。特异性试验表明,与新城疫病毒(NDV)、传染性支气管炎病毒(IBV)、减蛋综合征病毒(EDSV)、禽流感病毒H9N2亚型的阳性血清均无交叉反应；重复性试验结果表明,间接ELISA方法批内、批间变异系数小于10%。将本实验建立的方法与IDEXX公司ELISA检测试剂盒进行了比较,检测200份IBDV血清样品,阳性符合率为92.9%,阴性符合率100%。实验结果表明,本实验建立的IBDV抗体ELISA检测方法具有良好的特异性与重复性。
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.
引文
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