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.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
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)
作者简介
作者简介：程朝飞（1987-），男，河南登封人，硕士，主要从事动物重大疫病的防控与诊断，E—mail：donwuyixue217@163．com，Tel,010—62813876；
宫苗苗（1985-），女，山东泰安人，硕士，主要从事预防兽医学研究。
通讯作者：李刚，教授，E—mail：gli358@Gmail．com
