猪伪狂犬病病毒gE蛋白原核表达及野毒抗体间接ELISA检测方法的建立与应用

Development and Application of Indirect ELISA for Detection of Antibodies Against PRV and Prokaryotic Expression of gE Protein

为了建立一种简单、快速的猪伪狂犬病病毒(PRV)野毒抗体检测方法,试验采用原核表达技术对PRV流行毒株的gE蛋白进行了原核表达,以表达的重组蛋白作为包被抗原建立了检测PRV野毒抗体的间接ELISA检测方法。结果表明:获得了以包涵体形式表达的重组gE蛋白,经Western blot鉴定表明重组gE蛋白具有良好的免疫学活性,以该蛋白作为包被抗原建立的检测PRV野毒抗体的间接ELISA方法检测PRV疫苗免疫血清、CSFV、PRRSV、PCV-2、PPV、PEDV、FMDV阳性血清均为阴性,检测PRV野毒阳性血清的最低检出效价为1∶10240,批内和批间重复性试验的变异系数分别为2.19%~3.33%和3.01%~4.40%,与国外商品化gE-ELISA试剂盒的符合率达98.72%,应用该ELISA检测河南省部分地区采集的1128份猪血清样品,PRV野毒抗体阳性率为8.07%。说明建立的PRV野毒抗体间接ELISA检测方法具有良好的特异性、敏感性、重复性和临床适用性,为PRV野毒抗体的流行病学监测和净化提供了技术支持。

In order to develop a simple and rapid method for the detection of antibodies against Pseudorabies virus(PRV),the gE protein was expressed by prokaryotic expression technology and the expressed recombinant protein was used as the coating antigen for an indirect ELISA method.The results showed that the recombinant gE protein expressed in the form of inclusion body and had good immunological activity.The indirect ELISA was then developed for detection of PRV wild-type antibodies.The positive sera of PRV vaccine,CSFV,PRRSV,PCV-2,PPV,PEDV and FMDV were all negative.The lowest detectable dilution of PRV wild virus positive serum was 1:10240.The coefficients of variation of intra and inter batch repeatability tests were 2.19%-3.33%and 3.01%-4.40%,and the coincidence rate with foreign commercial gE-ELISA kit was 98.72%.Total 1128 pig serum samples were collected from some areas of Henan province and tested for the positive rate at 8.07%.The results showed that the indirect ELISA method developed here had good specificity,sensitivity,repeatability and clinical applicability,which provided technical support for the epidemiological monitoring and eradication of PRV infection.