猪蓝耳病病毒抗体双抗原夹心ELISA方法的建立及初步应用

Establishment of a sandwich ELISA for detection of antibodies against porcine reproductive and respiratory syndrome virus

根据PRRSV VR-2332株序列设计了1对扩增PRRSV N蛋白基因的特异性引物,从PRRSV北方株感染细胞中提取总RNA,通过RT-PCR获得长约372 bp的N蛋白编码基因片段。将其克隆到pGEX-6p-1质粒,构建了原核表达载体pPRRS-N。重组基因在大肠杆菌中表达出相对分子质量约为41 000的融合蛋白,目的蛋白表达量约占菌体蛋白的28.5%。利用此重组融合N蛋白建立了一种检测PRRSV特异性抗体的双抗原夹心ELISA,并通过与商品化试剂盒的应用比较对本方法进行了系统评价。分析了来自北京、山东、河南、河北4省区13个养猪场的260份血清,结果表明,本方法的敏感性和特异性分别为93.5%和86.7%,与IDEXX PRRSV抗体检测试剂盒检测结果的符合率达到91.5%。

The nucleocapsid protein encoding gene of PRRSV Beijing strain was amplified by using a RT-PCR and was cloned into pGEX-6p-1 vector to generate an expressing plasmid pPRRS-N.The recombinant was transformed into and expressed in Escherichia coli BL21（DE3）.A sandwich ELISA was developed to detect PRRSV specific antibodies in the infected sera,using the recombinant protein as coating antigen and the horseradish peroxidase-labelled recombinant protein as the tracer.Comparison between sandwich ELISA and IDEXX-ELISA for detecting 260 sera samples from 13 herds with different disorders,showed 91.5% agreement,indicating that the sandwich ELISA was specific and sensitive.