猪A群轮状病毒VP6的表达及IgG抗体ELISA检测方法的建立与初步应用  

Expression of VP6 and Development of An ELISA for Detection of IgG Antibodies against Porcine Rotavirus Group A

作　　者：陈淑华[1,2] 库旭钢[1,2] 闫贵伟 丁振江[1,2] 何启盖[1,2] 

机构地区：[1]农业微生物学国家重点实验室,湖北武汉430070 [2]华中农业大学动物医学院,湖北武汉430070

出　　处：《动物医学进展》2014年第7期1-6,共6页Progress In Veterinary Medicine

基　　金：国家生猪产业技术体系项目(CARS-36)

摘　　要：为建立猪A群轮状病毒抗体间接ELISA检测方法,用于开展对该病毒感染的流行病学调查和免疫抗体评估,进行了猪A群轮状病毒TM-a株内衣壳VP6基因的表达,用SDS-PAGE和Western blot鉴定分析。目的蛋白得到表达,大小约70ku,Western blot表明,该蛋白与猪A群轮状病毒阳性血清反应。用该蛋白作为包被抗原,通过对条件优化,包被抗原浓度为2μg/mL,待检血清稀释倍数为1∶40倍,标记抗体的稀释倍数为1∶4 000倍,建立了检测猪轮状病毒血清IgG抗体的间接ELISA方法。用该方法检测临床样品142份,并与间接免疫荧光试验(IFA)对比,结果显示,两者阳性符合率为94.6%,阴性符合率为83.4%,总符合率为91.5%,表明该方法有效可行。用建立的方法检测临床上不同年龄阶段的猪血清639份,分析其临床感染情况。结果证明该方法可用于猪轮状病毒流行病学调查、猪群抗体水平评估。The aim of this research was to establish an indirect ELISA method for clinically detection of antibodies against porcine rotavirus Group A（PoRV）in pigs.VP6 protein of Group A PoRV TM-a strain was successfully expressed in vitro.This recombinant protein was confirmed through SDS-PAGE and Western blot.The size of recombinant protein was 70 ku and can be recognized by positive serum against PoRV.After optimization,2μg/mL of the protein was used to coat the plate,the dilution of clinical serum and HRP-conjugated antibody were 1∶40and 1∶4 000,respectively.The indirect ELASA detection method was established and used to detect clinical samples.Totally,142 clinical samples were detected as positive according to our method and confirmed through indirect immunofluorescence assay（IFA）.The agreement of negative and positive were 83.4%,94.6%,resulting the general agreement of 91.5%between ELISA and IFA.This ELISA was further used to detect 638 serum samples collected from different ages of swine to analyse the infection rate of PoRV in swine farm.The developed ELISA could be a convenient and accurate method for the epidemiological investigation of PoRV infection in pigs.

关 键 词：猪轮状病毒 内衣壳蛋白 间接ELISA 

分 类 号：S852.659.4[农业科学—基础兽医学] Q789[农业科学—兽医学]