狂犬病病毒糖蛋白在大肠埃希菌中的表达及鉴定

目的表达狂犬病病毒糖蛋白（GP）,用于狂犬病疫苗免疫抗体评估和狂犬病病毒糖蛋白功能的研究。方法采用分析软件,分析其可能的抗原表位,利用PCR方法扩增狂犬病病毒SRV9疫苗株G蛋白抗原位点区域基因,PCR产物经EcoRI和SalI双酶切后,插入大肠埃希菌表达载体pGEX-6P-1,构建重组表达质粒pGEX-6P-1/G87a和pGEX-6P-1/G100a。将重组质粒转化大肠埃希菌BL21感受态细胞中,在IPTG诱导下表达目的蛋白,进行SDS-PAGE分析。表达蛋白进行电洗脱纯化和Western blot鉴定分析。结果成功构建了pGEX-6P-1/G87a和pGEX-6P-1/G100a表达质粒,序列分析表明,插入片段大小分别为1314 bp和1275 bp。SDS-PAGE分析结果证明,在大肠埃希菌系统中成功表达了狂犬病病毒部分糖蛋白,表达的融合蛋白含有GST标签,大小分别约为74×103和73×103。Western blot鉴定结果表明,表达产物有抗原特异性并能与狂犬病病毒抗血清反应。结论利用大肠埃希菌表达系统成功表达了狂犬病病毒部分糖蛋白,表达产物有良好的反应原性。

Expression and Identification of Rabies Virus Glycoprotein in Escherichia coli

Objective To express the glycoprotein（GP） of rabies virus（RV） as an antigen for evaluation of immune antibody in rabies vaccine.It could be useful for the further study of RV GP.Methods The genes of GP of rabies virus SRV_9 strain were analyzed.The fragments of genes of GP were amplified by PCR.The PCR products were digested with EcoRI and SalI and inserted into prokaryotic expression vector pGEX-6P-1 to construct the recombinant expression plasmids pGEX-6P-1/G87a and pGEX-6P-1/G100a.The recombinant plasmids were transferred into E.coli strain BL21（DE3）.The expression of proteins was induced by IPTG and analyzed by SDS-PAGE.The expressed proteins were purified by electroelution and identified by Western blotting.Results The recombinant expression plasmids pGEX-6P-1/G87a and pGEX-6P-1/G100a were constructed successfully and the size of insert fragments were 1314 bp and 1275 bp,respectively,identified by sequence analysis.SDS-PAGE analysis demonstrated that the recombinant proteins were expressed successfully in E.coli BL21 with the molecular weight of 74×103 and 73×103 with a GST-tag,respectively.Western blot analysis demonstrated that the recombinant proteins presented antigenic specificity and were reactive with RV antiserum.Conclusion It suggests that rabies virus glycoprotein can be expressed successfully in E.coli BL21 cells and they possess favorable immunoreactive activity.