基于猪非典型瘟病毒E2蛋白的间接ELISA检测方法的建立  

Establishment of an indirect ELISA for the detection of atypical porcine pestivirus based on E2 protein of APPV

作　　者：黎振标 许古明[1] 刘健新[1] 任旭皎 鲁荣 李慧子 张彭涛 宁章勇[1] LI Zhen-biao;XU Gu-ming;LIU Jian-xin;REN Xu-jiao;LU Rong;LI Hui-zi;ZHANG Peng-tao;NING Zhang-yong(College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学兽医学院

出　　处：《中国兽医科学》2019年第1期1-6,共6页Chinese Veterinary Science

基　　金：广东省优秀青年教师资助计划项目(Yq2013025)

摘　　要：猪非典型瘟病(APPV)是新近发现能引起仔猪先天震颤的病原体,血清学检测方法亟待构建。本研究制备了APPV原核表达的E2重组蛋白,以其作为包被抗原,建立了间接ELISA检测方法。结果表明,最佳抗原包被浓度为10μg/mL,最佳血清稀释度为1:40,阴阳性临界值D450=0.243,灵敏度达到1:64;与猪瘟、猪伪狂犬病、猪繁殖与呼吸综合征、猪流感及圆环病毒病的阳性血清均不发生交叉反应,特异性好;批内变异系数最大值为3.797%,批间变异系数最大值为5.425%,重复性好。利用建立的间接ELISA方法对50份猪临床血清样本进行检测,阳性率为12%。本研究基于E2蛋白建立的间接ELISA方法为猪非典型瘟病毒感染的检测提供了可靠的工具,也为开展APPV相关的研究工作提供了基础材料。Atypical porcine pestivirus (APPV)is a newly discovered pathogen which cause the congenital tremor of piglets,and the serological detection method needs to be established urgently.E2 protein is the major antigen of neutralizing antibodies against APPV and one of the most significant target of serological detection.In this study,the recombinant E2 protein of prokaryotic expression was coated as the antigen to establish an indirect ELISA method.In result,the optimal antigen coating concentration was 10μg/mL,and the optimal serum dilution was 1/40.The positive and negative critical value was D450=0.243 and the sensitivity of the indirect ELISA was 1/64.There were no cross reactions with the positive sera with classical swine fever virus,porcine pseudorabies virus,porcine reproductive and respiratory syndrome virus,swine influenza virus and porcinecircovirus virus, respectively,showing that it had a good specificity of detection.The coefficient of maximum variation was 3.797% and 5.425% in intra-and inter-assay,respectively,showing that the detection method was stable.The established indirect ELISA method was used to detect 50 different clinical porcine sera,and the positive rate was 12%.In conclusion,the establishment of the indirect ELISA method based on E2 protein provides a reliable tool for the detection of APPV infections,and also offers basic materials for developing APPV-related work.

关 键 词：猪非典型瘟病毒 E2蛋白 原核表达 间接ELISA 

分 类 号：S852.659.6[农业科学—基础兽医学]