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

作　　者：仝利剑[1,2,3] 赵建增 张社民 高荣[1] 高英杰[1] 慕泽鑫[1,2,3] 邢海云[1] 罗胜军[4] 温涛[1,2] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]北京必威安泰生物科技有限公司,北京100176 [3]山西省芮城县家畜家禽疫病防治站,山西芮城044600 [4]广东省农业科学院兽医研究所,广东广州510640

出　　处：《中国兽医学报》2010年第10期1273-1276,共4页Chinese Journal of Veterinary Science

基　　金：山西省科技攻关计划基金资助项目(20080311036-2)

摘　　要：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.

关 键 词：PRRSV N蛋白 双抗原夹心ELISA 

分 类 号：S852.65[农业科学—基础兽医学]