绿色荧光蛋白标记HIV融合蛋白基因表达载体

Construction of prokaryotic expression vector contains GFP-HIV(ENV-6C) fusion protein gene and its expression

目的利用基因工程技术,构建携带绿色荧光蛋白(green fluorescent protein,GFP)标记的HIV(ENV-6C)基因的表达载体,并在E.coliBL21中高效表达。方法经核酸内切酶酶切、连接,构建重组表达载体pRSETB-HIV(ENV-6C)-GFP,通过十二烷基磺酸钠-聚丙稀酰胺凝胶电泳(SDS-PAGE)、Western印迹杂交技术(Western blot)分析重组结果,转化到E.coliBL21中,荧光分光光度计检测含有重组质粒的大肠埃希菌培养物中重组蛋白的表达情况。结果成功构建重组原核表达载体pRSETB-HIV(ENV-6C)-GFP,并在E.coliBL21中实现高效表达,检则到发绿色荧光的融合蛋白GFP-HIV(ENV-6C)。结论融合蛋白具有人类免疫缺陷病毒(HIV)外壳蛋白的抗原活性,可用绿色荧光蛋白标记抗原,对制备HIV抗体及免疫诊断新技术有一定价值。

Objective To construct the prokaryotic expression vector pRSETB-HIV（ENV-6C）-GFP contains GFP-HIV （ENV-6C） fusion protein gene by using technology of genetic engineerng, and effectively express in E. coli BL21. Methods The recobmbination prokaryotic expression vector pRSETB-HIV（ENV-6C）-GFP was constructed through restricted digestion and ligatiln by restriction enzymes and DNA ligase. It was identified by the restriction enzyme analysis and the Western blot analysis. And then the expression was transfected into E. coli BL21. Green fluorescent emanated by the recombinant protein in the E. coil BL21 culture was observed under fluorescent microscope. Results The prokaryotic expression vector pRSETB-HIV（ENV-6C）-GFP was successful reconstructed and effectively expressed. The recombinant protein could be detected through its green fluorescent. Conclusion The recombinant protein pRSETB-HIV（ENV-6C） also has antigenicity of HIV coat protein. The antigen of HIV（ENV-6C） protein is marked by the green fluorescent protein, it can establish the theoretical foundation of preparation antibody of HIV and immunodiagnostic new techniques.