猪流行性腹泻病毒S2蛋白间接ELISA检测方法的建立与应用  

Establishment and application of an indirect ELISA antibody detection method for S2 protein of porcine epidemic diarrhea virus

作　　者：王亚涛 陶洁[2,3,4] 李本强[2,3,4] 程靖华 石迎[2,3,4] 刘惠莉 WANG Ya-tao;TAO Jie;LI Ben-qiang;CHENG Jing-hua;SHI Ying;LIU Hui-li(National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306,China;Institute of Animal Science and Veterinary Medicine,Shanghai Academy of Agricultural Sciences,Shanghai 201106,China;Shanghai Engineering Research Center of Breeding Pig,Shanghai 201302,China;Division of Animal Genetic Engineering,Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding,Shanghai 201106,China)

机构地区：[1]上海海洋大学水产科学国家级实验教学示范中心,上海201306 [2]上海市农业科学院畜牧兽医研究所,上海201106 [3]上海种猪工程技术研究中心,上海201302 [4]上海市农业遗传育种重点实验室,上海201106

出　　处：《中国兽医科学》2021年第3期269-274,共6页Chinese Veterinary Science

基　　金：上海市科技兴农推广项目[沪农科推字(2017)第1-11号]。

摘　　要：本研究基于猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)S2截短蛋白,建立了特异性Ig G抗体的间接ELISA检测方法,用于临床PEDV特异性抗体水平的监测。前期筛选了富含中和表位的S2截短基因,体外表达后作为ELISA包被抗原,用于捕获PEDV Ig G抗体,以山羊抗猪HRP-Ig G作为二抗,通过条件优化,建立了检测猪血清中PEDV Ig G抗体的间接ELISA方法。最佳反应条件为:S2抗原蛋白的最佳包被浓度为2μg/m L,临床血清样品和酶标二抗的最佳稀释倍数分别为1∶400和1∶20000。该方法的批内和批间重复变异系数(CV)分别为1.81%~8.52%和1.18%~7.41%,重复性较好;该方法检测猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)及牛病毒性腹泻病毒(BVDV)的阳性血清时结果均为阴性,特异性较强。该方法与商品化PEDV抗体检测试剂盒相比较,阳性符合率为95.18%,阴性符合率为91.43%,说明该方法可用于临床血清抗体筛查。进一步利用此方法对324份猪的临床血清样品进行检测,发现PEDV抗体阳性血清占62.96%,与临床现状基本吻合。上述结果表明,该方法适用于临床猪群中PEDV Ig G抗体水平普查,为PEDV的疫病防控及灭活疫苗评价提供了有效的技术支持。Based on the truncated S2 protein of porcine epidemic diarrhea virus(PEDV),an indirect ELISA method was established for detection of the specific Ig G antibody in clinical practice.The truncated S2 gene rich in neutralizing epitopes was screened and expressed in vitro as an ELISA coating antigen to capture PEDV Ig G antibody.Goat anti-porcine HRP Ig G was used as the secondary antibody.The indirect ELISA method for detecting PEDV Ig G antibody in pig serum was established by optimizing reaction conditions.The optimal reaction conditions were as follows:the best coating concentration of S2 antigen protein was 2μg/m L,and the optimal dilution ratios of clinical serum samples and enzyme labeled secondary antibody were 1∶400 and 1∶20000,respectively.Repetitive experiments were showed the coefficient of variation within-run ranged from 1.81%to 8.52%and that between-runs ranged from1.18%to 7.41%,with good repeatability.Moreover,PRV,PCV2,CSFV,and BVDV positive serum samples were detected by this method and showed negative results,which showed strong specificity.Compared with the commercial PEDV antibody detection kit,the positive coincidence rate was 95.18%and the negative coincidence rate was 91.43%.It showed that the method can be used in clinical serum antibody screening.Further,this method was used to detect 324 pig clinical serum samples.The results indicated that PEDV antibody positive serum accounted for 62.96%,which was basically consistent with the clinical situation.Therefore,this method is suitable for the general survey for PEDV Ig G antibody level in clinic,and provides effective technical support for the prevention and control of PEDV and the evaluation of inactivated vaccine.

关 键 词：猪流行性腹泻病毒 间接ELISA S2截短蛋白 IGG 

分 类 号：S852.659.6[农业科学—基础兽医学]