猪繁殖与呼吸综合征病毒Nsp12的原核表达及间接ELISA方法的建立

Prokaryotic Expression of PRRSV Nsp12 and Establishment of Indirect ELISA

Nsp12作为猪繁殖与呼吸障碍综合征病毒(PRRSV)非结构蛋白,其诱导产生的抗体能作为检测野毒感染的良好指标。用RT-PCR方法获得PRRSV Nsp12的基因,用原核表达系统pET-32a(+)在BL21(DE3)细胞中表达Nsp12,通过亲和层析纯化蛋白。用表达的Nsp12建立检测PRRSV抗体的间接ELISA方法。结果显示,获得了Nsp12基因及原核表达Nsp12,建立了检测抗Nsp12抗体的间接ELISA方法,阴性、阳性临界值为OD 450 nm值为0.227,92份血清样品的检测结果表明建立的ELISA方法特异性较好,为猪场监测PRRSV野毒感染提供了技术支持。

Nsp12 is a non-structural protein of PRRSV,and its induced antibodies can be used as a good indicator for detecting wild virus infection.In this study,the Nsp12 gene,which is an non-structural protein of PRRSV,was obtained by RT-PCR method.The prokaryotic expression system pET-32a(+)was used to express Nsp12 protein in BL21(DE3)cells.The protein was purified by affinity chromatography.An indirect ELISA method for detecting PRRSV antibodies was established using the expressed Nsp12 protein.As a result,Nsp12 gene was successfully obtained and Nsp12 protein was successfully expressed.We established an indirect ELISA method for detecting anti-Nsp12 antibodies,and determined that the cut-off value for negative and positive was OD 450 nm 0.227.The detection results of 92 serum samples showed that the established ELISA method was more specific.The indirect ELISA method for detecting PRRSV Nsp12 antibodies established in this study provides technical support for the monitoring of PRRSV wild virus infection in pig farms.