丙型肝炎病毒核心蛋白与NS4B融合蛋白的构建和表达

Construction of fusion vector of core gene and NS4B gene of HCV and its expression in E. coli

目的:构建丙型肝炎病毒(HCV)核心蛋白(core)与NS4B融合表达克隆,并在大肠埃希菌中表达。方法:应用PCR方法从抗HCV阳性血清中扩增出HCV核心蛋白基因和NS4B基因的c DNA片段,将两部分基因片段拼接成一条完整c DNA,连接到表达载体p ET28a上,菌落PCR和测序方法选择阳性核心蛋白-NS4B-p ET28a重组质粒。将阳性重组质粒转化到大肠埃希菌BL21中进行表达,分别用SDS-PAGE和蛋白质印迹分析表达的重组融合蛋白。结果:琼脂糖凝胶电泳结果显示,成功获得核心蛋白基因和NS4B基因,并成功拼接成一条完整c DNA;菌落PCR和测序结果表明重组融合克隆核心蛋白-NS4B-p ET28a构建成功。SDS-PAGE结果表明重组融合质粒在大肠埃希菌BL21中成功表达,蛋白质印迹结果显示表达的重组蛋白与抗HCV阳性血清具有反应性。结论:成功构建了HCV核心蛋白与NS4B的重组融合克隆,并成功在大肠埃希菌中表达。

Objective: To construct a fusion gene clone of hepatitis C virus( HCV) core protein and NS4 B and to check it expression in E. coli. Methods: c DNA fragments of HCV core gene and NS4 B gene were separately amplified from the anti-HCV positive sera by PCR,followed by connecting them to form an intact c DNA and inserted into the expression vector p ET28 a. Positive recombinant plasmid named coreNS4 B-p ET28 a was screened and confirmed by colony PCR and DNA sequencing. Afterwards it was transformed into E. coli BL21 to express recombinant fusion protein,which was analyzed by SDS-PAGE and Western blotting. Results: Agarose gel electrophoresis showed that core gene and NS4 B gene as well as the corresponding c DNA consisting of them were obtained successfully. Colony PCR and DNA sequencing showed that the plasmid core-NS4 B-p ET28 a was constructed successfully. SDS-PAGE clearly demonstrated the successful expression of the fusion protein in E. coli BL21. Western blotting showed that the protein had reactivity with anti-HCV positive sera. Conclusion: The fusion expression vector for core gene and NS4 B gene of HCV was successfully constructed and the protein was expressed successfully in E. coli.