检测猪瘟病毒E0抗体间接ELISA方法的建立及其初步应用  

Establishment of an indirect ELISA for detection of antibodies against classical swine fever virus based on CSFV-E0 antigens

作　　者：王旭 蔡梦露 马振乾 陈洁 龚文杰 常晓冉 张泽财 涂长春 王新平 WANG Xu;CAI Meng-lu;MA Zhen-qian;CHEN Jie;GONG Wen-jie;CHANG Xiao-ran;ZHANG Ze-cai;TU Chang-chun;WANG Xin-ping(Ministry of Education Key Laboratory of Zoonosis Research/College of Animal Medicine,Jilin University,Changchun 130062,China;Qingdao Yibang Bioengineering Co.,Ltd.,Qingdao,Shandong 266032,China;Military Veterinary Institute,Academy of Military Medical Sciences,Changchun 130122,China)

机构地区：[1]吉林大学动物医学学院/教育部人兽共患病研究重点实验室,吉林长春130062 [2]青岛易邦生物工程有限公司,山东青岛266032 [3]军事医学研究院军事兽医研究所,吉林长春130122

出　　处：《中国兽医学报》2020年第3期441-449,456,共10页Chinese Journal of Veterinary Science

基　　金：国家“十三五”重点研发计划资助项目(2016YFD0500904,2017YFD0500104)。

摘　　要：应用RT-PCR技术扩增了猪瘟病毒的E0基因片段,并将其分别克隆到pGEX-4T-1与pET-28a载体,获得了pGEX-4T-1-E0与pET-28a-E0重组表达质粒。以IPTG诱导表达的纯化GST-E0和His-E0重组蛋白为包被抗原,建立检测猪瘟病毒E0抗体的间接ELISA方法。采用方阵滴定法,确定出GST-E0抗原和His-E0抗原的最佳包被量分别为50ng/孔和100ng/孔,血清最佳稀释度为160倍。对80份猪瘟阴性血清样品进行了检测与统计学分析,确定出GST-E0和His-E0重组蛋白的间接ELISA方法的临界值分别为0.167和0.176。特异性、敏感性和重复性试验结果表明,本试验建立的基于GST-E0和His-E0蛋白的间接ELISA方法均具有特异、敏感和重复性好等优点。应用GST-E0和His-E0的间接ELISA方法及IDEXX E2抗体检测试剂盒平行检测88份猪血清样品,结果显示基于GST-E0和His-E0的间接ELISA方法与IDEXX试剂盒的符合率分别为78.41%和84.09%。以猪瘟病毒接毒细胞为抗原,应用免疫荧光试验(IFA)对His-E0间接ELISA和IDEXX检测试剂盒检出的阴性与阳性血清样品进行了验证,结果IFA与His-E0间接ELISA方法的符合率为93.18%;IFA与IDEXX试剂盒的符合率为90.91%,表明本试验所建立的基于His-E0的间接ELISA方法用于检测猪瘟抗体优于IDEXX公司检测E2抗体的试剂盒。该方法为鉴别E2亚单位疫苗免疫后的猪群中是否存在野毒感染提供了技术手段。Based on the nucleotide sequence of the classical swine fever virus(CSFV)in GenBank,the primers for E0gene were designed and used to amplify E0gene fragment.After the fragment was cloned into pGEX-4T-1and pET-28avectors,the prokaryotic expression plasmids pGEX-4T-1-E0 and pET-28a-E0were generated,respectively,and recombinant E0protein expressions were induced by IPTG.The purified GST-E0and His-E0recombinant proteins were used as antigens for the establishment of the indirect ELISA to detect the antibodies against CSFV.The concentrations of the coating antigens for GST-E0and His-E0were optimized by square matrix titration to be 50ng/well and 100ng/well,respectively,with a serum dilution in 160×.By statistical analysis of 80CSFV-negative serum samples,the thresholds for indirect ELISA based on GST-E0and His-E0 were defined as 0.167and 0.176,respectively.Results from the specificity,sensitivity and reproducibility tests showed that the established indirect ELISA methods were specific,sensitive and reproducible.Detection of the 80serum samples using GST-E0,His-E0ELISA and IDEXX E2 detection kit showed the coincidence rates of the indirect ELISA method based on GST-E0and His-E0recombinant proteins were 78.41% and 84.09%,respectively,with that of IDEXX E2detection kit.The indirect ELISA method of His-E0is more consistent than the indirect ELISA method based on GST-E0.To validate the above results,immunofluorescent assay was employed to detect the negative and positive serum samples using CSFV-infected cells as antigen.The results showed that the coincidence rates of IFA assay with His-E0ELISA and IDEXX E2detection kit were 93.18% and 90.91%,respectively,indicating the consistency and advantage of His-E0ELISA method over the IDEXX E2detection kit.Thus,it provides a pragmatic method to differentiate the antibody elicited by wild strain from E2subunit vaccine that is widely used currently.

关 键 词：猪瘟病毒 E0基因 间接ELISA 符合率比较 

分 类 号：S852.65[农业科学—基础兽医学]