伪狂犬病病毒变异株gC抗体间接ELISA检测方法的建立及初步应用

Establishment and Preliminary Application of Indirect ELISA for Detection of Variant Pseudorabies Virus gC Antibody

本研究旨在建立变异伪狂犬病病毒FJ-2012株gC蛋白抗体间接ELISA检测方法,掌握不同猪场猪群的gC抗体水平情况。将FJ-2012株gC基因连接至pCzn1载体,转染至Arctic express(DE3)感受态细胞,IPTG诱导表达产物经SDS-PAGE和Western blot验证,通过矩阵法试验、临界值的确定、特异性试验、重复性和敏感性试验,建立PRV-gC抗体间接ELISA检测方法,并对源于不同猪场的280份血清进行PRV-gC抗体检测。结果表明,成功表达获得FJ-2012株pCzn1-gC重组蛋白;建立的间接ELISA方法的最佳抗原包被浓度为10μg·mL^(-1)和最佳样品血清稀释比例为1∶50,阴性和阳性判定的OD_(650 nm)临界值为0.406~0.438,介于之间的判定为可疑;临床检测结果显示,120份盲样猪血清PRV-gC抗体阳性率为91.67%、PRV-gB抗体阳性率为95.00%,两种抗体检测结果的整体符合率为95.83%;PR不稳定的猪场血样PRV-gC抗体阳性率为96.25%(77/80),而PRV已净化的种猪场血样PRV-gC抗体阳性率为78.75%(63/80)。因此,建立的PRV变异株gC抗体间接ELISA检测方法为临床猪血清抗体检测提供了特异、灵敏和稳定的技术,也为开展变异PRV血清学调查奠定基础。

The purpose of this study was to establish an indirect ELISA method for the detection of gC protein antibody of new pseudorabies virus FJ-2012 strain,and then the method was used to detect the level of gC antibody in different pig farms.The gC of FJ-2012 strain was connected to pCzn1 vector,then the recombinant vector was transfected into Arctic express(DE3)competent cells.The expression product induced by IPTG was verified by SDS-PAGE and Western blot.The indirect ELISA method for detecting PRV gC antibody was established by matrix test,determination of critical value,specificity test,repeatability,and sensitivity test.two hundred and eighty sera samples from different pig farms were detected for PRV gC antibody.The results showed that the pCzn1 gC recombinant protein of FJ-2012 strain was successfully expressed in Arctic express(DE3).The optimal antigen coating concentration of the indirect ELISA method was 10μg·mL^(-1),and the optimal serum dilution ratio of sample was 1∶50.The critical value of OD_(650 nm) for negative and positive judgment were 0.406 and 0.438,and the critical value between 0.406-0.438 were judged as suspicious.Clinical test results showed that,the positive rates of PRV gC antibody and PRV gB antibody in 120 blind pig serum were 91.67%and 95.00%respectively.The general coincidence rate of these two antibodies testing results were 95.83%.The positive rate of PRV gC antibody in blood samples from pig farms with unstable PR was 96.25%(77/80),however the positive rate of PRV gC antibody in blood samples from pig farms with purified PRV was 78.75%(63/80).Therefore,the indirect ELISA method established in this study not only provides a specific,sensitive and stable tool for the detection of porcine serum antibody,but also lays a foundation for the serological investigation of variant PRV.