狂犬病病毒G基因的克隆表达及间接ELISA检测方法的初步建立与应用  

Establishment of indirect ELISA and its application based on expressed G protein of rabies virus

作　　者：王净[1,2] 李刚[1] 史利军[1] 邱文英[1] 曾妮[1] 穆秀明[2] 高志花[2] 王慧文[2] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]河北北方学院动物科技学院,张家口075131

出　　处：《中国人兽共患病学报》2011年第4期279-282,共4页Chinese Journal of Zoonoses

基　　金：中央级公益性科研院所基本科研业务费专项资金(2009QN-3;2010JS-2;0032007008);北京市科委基金项目(Z07010501780701);国家农业行业公益项目(2008-3)联合资助

摘　　要：目的以重组G蛋白为检测抗原,应用间接ELISA方法检测狂犬病病毒抗体。方法根据GenBank公布的狂犬病病毒LEP-Flury株基因序列设计引物,PCR扩增出目的基因,连接pGM-T载体,重组质粒经BamH I和XhoⅠ双酶切,连接到表达载体pGEX-6P-1,构建重组表达载体pGEX-G。将重组表达载体转化大肠杆菌BL21(DE3)感受态细胞中,在IPTG诱导下表达目的蛋白,表达蛋白纯化后SDS-PAGE电泳分析并经Western blotting鉴定。纯化的G蛋白作为包被抗原,建立间接ELISA方法检测93份犬血清抗体。结果成功构建了克隆载体pGM-G和表达载体pGEX-G,高效表达了狂犬病病毒G蛋白,融合蛋白分子量大小为79kDa,表达蛋白可与狂犬病病毒抗血清发生特异性反应。建立的检测方法灵敏度达到1∶1 600,与商品化的试剂盒相比符合率为96.8%。结论成功表达狂犬病病毒G蛋白,表达产物可作为检测抗原用于间接ELISA方法中狂犬病病毒抗体的检测。To establish indirect ELISA and detect canine sera,prokaryotical glycoprotein was expressed from rabies virus in this study.In what follows,the complete length of G gene from rabies virus was amplified by PCR using a pair of specific primers designed according to the relevant sequences from GenBank,and the PCR product was cloned into vector pGM-T.The recombinant vector was digested with BamH I and XhoⅠand inserted into the prokaryotic expression vector pGEX-6P-1 to obtain the recombinant expressed vector pGEX-G and it was transferred into the E.coli strain BL21（DE3） cells.The expression of proteins was induced by IPTG and these proteins were purified and analyzed by SDS-PAGE.And they were identified by Western blotting and the size of insert fragments was 79kDA,which could be specifically reacted with RV antiserum.The indirect ELISA assay using the purified glycoprotein as the antigen was applied in detecting 93 canine serum samples.The sensitivity of the ELISA was 1∶1 600.Comparing with the commercial ELISA kit,the accuracy of the ELISA was 96.8%.

关 键 词：狂犬病病毒 G蛋白 表达 间接ELISA 

分 类 号：R373.9[医药卫生—病原生物学]