基于H蛋白表位合成肽的小反刍兽疫病毒抗体间接ELISA检测方法的建立  

Establishment of an iELISA Method for Detection of Antibody againist Peste des Petits Ruminants Virus Based on H Protein Epitope Peptide

作　　者：钱榜 李彦敏[1,2] 朱学亮 张学燕[1] Niyokwishimira Alfred 窦永喜 张志东[1,2] QIAN Bang;LI Yanmin;ZHU Xueliang;ZHANG Xueyan;Niyokwishimira Alfred;DOU Yongxi;ZHANG Zhidong(State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;College of Animal Husbandry and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,China)

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,兰州730046 [2]西南民族大学畜牧兽医学院,成都610041

出　　处：《畜牧兽医学报》2021年第1期144-153,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点研发计划项目(2016YFD0500108,2016YFD0500901);国家绒毛用羊产业技术体系建设专项资金(CARS-39-13);中央级公益性科研院所基本科研业务费专项(Y2020GH19)。

摘　　要：本研究旨在建立针对小反刍兽疫病毒(peste des petits ruminants virus, PPRV)H蛋白抗体的iELISA检测方法。以筛选的H蛋白B细胞表位为基础,选择反应原性较好的4个抗原表位肽串联后合成作为包被抗原,优化各步反应条件及筛选各种缓冲液,并确定检测临界值。经优化后,多表位抗原肽的最佳包被量为1.5×10~(-6)μg·孔~(-1),血清临界稀释度为1∶100,酶标二抗最佳稀释度为1∶80 000;包被液为碳酸氢盐缓冲液,血清和二抗稀释液均为PBST溶液,封闭液为2%BSA溶液;阴、阳性临界值为0.25。不同血清的检测结果表明,批内及批间检测结果变异系数均小于10%,证明该方法具有良好的稳定性;对羊痘、口蹄疫、蓝舌病阳性血清的检测均为阴性,说明该方法具有良好的特异性;与ID-Vet公司的cELISA试剂盒对306份临床血清平行检测,两者相对符合率为92.81%。该研究所建立的PPRV H蛋白抗体iELISA检测方法特异、稳定、敏感,操作比cELISA简便快捷,省时省力,可用于临床小反刍兽疫抗体检测。This study aims to establish an iELISA method for the detection of anti-PPRV H protein antibody. Four epitope peptides with good reactivity were selected and synthesized as the coating antigen based on the B cell epitopes of H protein. Optimization of the reaction conditions of each step and screening various buffers were performed and the cut-off value of detection was determined. After optimization, the optimal coating amount of multi-epitopes antigen peptide was 1.5×10 -6 μg·well -1 , the optimal working dilution of serum and the enzyme-labeled secondary antibody was 1:100 and 1:80 000, respectively. The carbonate-hydrogen buffer was chosen as the coating buffer, and PBST buffer was adopted as dilution buffer for the serum and the secondary antibody. The appropriate blocking buffer was determined to be 2% BSA solution and the cut-off value of the assay was 0.25. The test results of clinical sera showed that the coefficients of variation of intra- and inter-assay were less than 10%, which proved its good repeatability. When the assay was tested with the positive sera of sheep pox, FMD, and BT, there was no cross-reaction obtained, indicating that the assay was specific for the detection of the PPRV antibody. The detection results of 306 sera samples showed that the developed ELISA had a relative sensitivity of 92.81% when compared with the commercially available ID Vet cELISA Kit. This study has developed a specific, repeatable, and sensitive iELISA method for detecting antibodies of PPRV H protein, and it is more convenient, timesaving, and labor-saving than cELISA. Therefore, the assay can be used for the detection of antibodies against PPRV.

关 键 词：小反刍兽疫病毒 H蛋白 B细胞表位 IELISA 

分 类 号：S852.659.5[农业科学—基础兽医学]