基于重构NDRV σC 蛋白的间接ELISA抗体检测方法的建立  

Establishment of an indirect ELISA antibody detection method based on reconstructed NDRVσC protein

作　　者：武华 林伟东 孟利佳 阴雅洁 乔思娜 倪维玲 侯绍华[3] 徐倩倩 董世山[1,5] WU Hua;LIN Weidong;MENG Lijia;YIN Yajie;QIAO Sina;NI Weiling;HOU Shaohua;XU Qianqian;DONG Shishan(College of Veterinary Medicine,Agricultural University of Hebei,Hebei 0710ol,China;School of Medicine,Shanghai Jiaotong University,Shanghai 200025,China;Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 10o193,China;Hebei Chuangan Biotechnology Co.,Ltd.,Baoding,Hebei 071001,China;North China Scientific Observation and Experimental Station of Animal Pathogenic Biology,Ministry of Agriculture,Baoding,Hebei 071001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]上海交通大学医学院,上海200025 [3]中国农业科学院北京畜牧兽医研究所,北京100193 [4]河北创安生物科技有限公司,河北保定071001 [5]农业部动物疫病病原生物学华北科学观测实验站,河北保定071001

出　　处：《中国兽医学报》2022年第8期1557-1563,共7页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2017YFD0500806);河北省重点研发计划资助项目(20326622D)。

摘　　要：旨在建立灵敏快速诊断新型鸭呼肠孤病毒(novel duck reovirus,NDRV)抗体的间接ELISA(indirect ELISA,iELISA)方法,为水禽养殖业提供科学有效的检测手段。先前报道σC蛋白为沉淀表达,因此本研究利用大肠杆菌的密码子偏嗜性等规律优化其基因序列进行原核表达,纯化出高纯度的可溶性σC蛋白。采用方阵滴定法确定σC蛋白包被浓度和样品稀释度,初步建立NDRV的iELISA检测方法。分别对84份阳性和180份阴性质控血清进行分析,构建受试者工作特征曲线(ROC),通过Youden指数选择灵敏性和特异性最佳的P值作为阴阳临界值。使用质控阴、阳血清评价试剂盒的批内和批间重复性。用本研究的和实验室先前建立的全病毒包被的iELISA方法同时对质控血清进行检测,比较其符合率。通过方阵滴定法确定各组分条件为σC蛋白包被质量浓度0.5μg/孔、血清稀释度1∶320。构建的ROC曲线下面积为1.00,最佳阴阳临界值为0.076,在此临界值上的灵敏性和特异性均为100%。全病毒包被板和σC蛋白包被板符合率为100%,但前者检测样品的D_(450)值偏低,σC蛋白包被板则弥补了这一缺点。本研究为NDRV提供特异性和灵敏性良好的iELISA抗体检测方法。This study aims to establish a sensitive and rapid indirect ELISA(iELISA)method for detecting novel duck reovirus(NDRV)antibodies and provide a scientific and effective detection method.It was reported that theσC protein was expressed in the precipitation with the Escherichia coli expression system.Therefore,gene sequence was optimized for prokaryotic expression based on the codon preference and high-purity solubleσC protein was expressed and purified.Then the square matrix titration method was used to determine theσC protein coating concentration and sample dilution,and the iELISA detection method for NDRV was established.Then the negative and positive control serum were used to evaluate the intra-and inter-assay reproducibility of the kit.The control serums were tested simultaneously by using the whole virus-coated plate,and their coincidence rate was compared.The best conditions of each component determined by the square matrix titration method were as follows:theσC protein coating concentration was 0.5μg/well,and serum dilution ratio was 1∶320.Then 84positive and 180negative control serums were analyzed to construct a receiver operating characteristic curve(ROC)curve,and the P value with the best sensitivity and specificity was selected as the cutoff value through the Youden index.The area under the ROC curve constructed was 1.00,and the best cutoff value was 0.076.The sensitivity and specificity at this cutoff value were both 100%.The coincidence rate of the whole virus-coated plate and theσC protein-coated plate was 100%,but the D450value of the test sample of the former was low,and theσC protein-coated plate makes up for this shortcoming.This study provides an iELISA antibody detection method with good specificity and sensitivity for NDRV.

关 键 词：新型鸭呼肠孤病毒 原核表达 σC蛋白 间接ELISA方法 

分 类 号：S852.65[农业科学—基础兽医学]