基于狂犬病病毒重组蛋白的ELISA检测方法比较研究  

Comparative Study on the Indirect ELISA Methods Based on the Different Recombinant Proteins of Rabies Virus

作　　者：程朝飞[1] 宫苗苗[1] 田康乐[1] 王勇[1,2] 赵占中[1] 史利军[1] 李刚[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所动物营养学国家重点实验室,北京100193 [2]中国农业大学动物医学院,北京100193

出　　处：《畜牧兽医学报》2012年第12期1925-1930,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家高技术研究发展计划(863计划)(2008AA10Z411);北京市科委项目(Z07010501780701);国家公益行业项目(20083014;200903037-2)

摘　　要：原核表达狂犬病病毒的基质蛋白(M),并以此作为包被抗原建立间接ELISA检测方法,与以狂犬病病毒磷蛋白(P)作为包被抗原建立的间接ELISA方法进行比较。根据GenBank中公布的狂犬病病毒LEP-Flury株M基因序列设计特异性引物并引入SmaⅠ和NotⅠ酶切位点,经RT-PCR扩增得到目的基因,连接pCR 2.1载体,构建重组质粒pCR-RV-M,重组质粒用SmaⅠ和NotⅠ进行双酶切,酶切产物定向克隆至原核表达载体pGEX-6P-1中,构建重组表达载体pGEX-RV-M。将重组表达载体转化E.coli BL21(DE3)感受态细胞,使用IPTG诱导表达目的蛋白,SDS-PAGE分析表明蛋白表达量较大,且主要以可溶性的形式表达。亲和层析纯化目的蛋白,Westernblot表明融合蛋白具有良好的反应原性,使用纯化的融合蛋白作为包被抗原建立了间接ELISA方法并检测了95份血清,同时使用带有His标签的重组狂犬病病毒磷蛋白P(RV-His-P)建立的ELISA方法和商品化的ELISA试剂盒检测该血清。结果表明:与商品化的以全病毒作为包被抗原的ELISA检测试剂盒相比,使用重组P蛋白作为包被抗原建立的间接ELISA检测方法具有更高的符合率,能够代替全病毒作为诊断抗原建立检测方法。This study was designed to compare the indirect ELISA methods for detection of dog antibodies against rabies virus based on the recombinant matrix protein （M） and phosphoprotein （P）. The M gene of rabies virus LEP-Flury strain was amplified by PCR and cloned into prokary- otic expression vector pGEX-6P-1. The resultant constructed pGEX-RV-M plasmid was trans- formed into BL21 （DE3） and protein expression was analyzed by SDS-PAGE and Western blot. The results of SDS-PAGE showed that the M protein was efficiently expressed, which were mainly soluble, and purified with the affinity chromatography. The results of Western blot indi- cated that the recombinant protein M showed good immunogenicity. The indirect ELISA methodwas established detected by the spectively. The RV as antigen, dence rate, and with the purified recombinant protein M, and a total of 95 serum samples were method and the indirect ELISA method based on the recombinant protein P re- results showed that compared with the commercially available ELISA kit coating the indirect ELISA method based on recombinant protein P had a higher coinci- it can replace the ELISA method based on RV.

关 键 词：狂犬病病毒 基质蛋白M 原核表达 间接ELISA 磷蛋白P 

分 类 号：S855.3[农业科学—临床兽医学] S858.292[农业科学—兽医学]