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 Xho Ⅰ and 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 EL ISA was 1:1 600. Comparing with the commercial ELISA kit, the accuracy of the ELISA was 96.8％.
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