基于狂犬病病毒重组蛋白的ELISA检测方法比较研究

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Sma Ⅰ和NotⅠ酶切位点,经RT-PCR扩增得到目的基因,连接pCR(R)2.1载体,构建重组质粒pCR-RV-M,重组质粒用Sma Ⅰ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,Western blot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-l. The resultant constructed pGEX-RV-M plasmid was transformed 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 indicated that the recombinant protein M showed good immunogenicity. The indirect ELISA methodwas established with the purified recombinant protein M, and a total of 95 serum samples were detected by the method and the indirect ELISA method based on the recombinant protein P respectively. The results showed that compared with the commercially available ELISA kit coating RV as antigen, the indirect ELISA method based on recombinant protein P had a higher coincidence rate, and it can replace the ELISA method based on RV.