汉坦病毒包膜糖蛋白G2重组腺病毒的构建及其在Hela细胞中的表达

Construction of a recombinant adenovirus carrying the Hantaan virus glycoprotein G2 gene and its expression in Hela cells

摘要:

本研究旨在构建可表达汉坦病毒(HTNV)糖蛋白G2的重组腺病毒。应用PCR方法扩增G2编码基因, 经T/A克隆、测序鉴定后再亚克隆到腺病毒shuttle载体pAd5-CMV中并用磷酸钙沉淀法分别将携带G2编码基因的重组腺病毒shuttle载体与携带报告基因eGFP的腺病毒骨架质粒共转染HEK293细胞, 包装、扩增、纯化后得到携带HTNV糖蛋白G2编码基因的重组腺病毒; 用重组腺病毒感染Hela细胞并收获蛋白, 间接免疫荧光、Western blotting检测蛋白表达。经酶切鉴定表明已成功构建了携带G2基因的重组腺病毒载体; RT-PCR鉴定表明目的基因能够在感染重组腺病毒的Hela细胞中转录; 荧光显微镜观察重组腺病毒感染的Hela细胞, 可见报告基因eGFP的表达; 间接免疫荧光法和Western blotting均证实表达产物可被抗G2单克隆抗体所识别, 表明糖蛋白G2在感染细胞中得到了表达。本研究成功构建了可表达HTNV包膜糖蛋白G2的重组腺病毒, 转染宿主细胞可稳定表达目的蛋白, 为HTNV糖蛋白G2的结晶、结构解析研究以及新型汉坦病毒疫苗的研制奠定了基础。

Abstract:

We constructed the recombinant adenovirus expressing the glycoprotein G2 of Hantaan virus. Firstly we obtained coding gene fragment of G2 by PCR, and subsequently inserted the gene of interest into the Adenoviral pShuttle vector pAd5-CMV. Then we co-transfected the recombinant pShuttle vector and adenovirus skeleton plasmid into HEK293 cells by Calcium phosphate precipitation method. After the recombinant adenovirus were packaged and amplified in HEK293 cells, we observed the expression of reporter gene eGFP by fluorescent microscopy, and we obtained the recombinant adenovirus containing Hantaan virus glycoprotein G2. The recombinant adenoviruses were used to infect Hela cells and the expressed protein was detected by Indirect Immuno-fluorescence and Western blotting. The construct was confirmed at several levels: first restriction enzyme analysis demonstrated that the recombinant adenovirus vector was constructed correctly, second RT-PCR showed that the G2 gene could transcribe correctly in Hela cells. Then Fluorescent microscopy proved the expression of eGFP in the infected Hela cells. Finally, Indirect Immuno-fluorescence and Western-blot confirmed the expression of interested protein identified by anti-G2 monoclonal antibody. In conclusions, this study successfully constructed the recombinant adenovirus containing Hantaan virus envelope glycoprotein G2, meanwhile obtained the G2 protein, it may lay solid foundation for the structure study of G2 protein and the new vaccine of Hantaan virus.