Huyan Ting, Xue Xiaoping, Song Kai, Wang Hua, Yang Hui, Wang Wei
Faculty of Life Science, Northwestern Ploytechnical University, Xi'an 710072, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Oct;25(10):1579-85.
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 Immune-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.
我们构建了表达汉滩病毒糖蛋白G2的重组腺病毒。首先，通过聚合酶链反应（PCR）获得G2的编码基因片段，随后将目的基因插入腺病毒穿梭载体pAd5-CMV中。然后，我们采用磷酸钙沉淀法将重组穿梭载体和腺病毒骨架质粒共转染至人胚肾293（HEK293）细胞中。在HEK293细胞中包装并扩增重组腺病毒后，我们通过荧光显微镜观察报告基因增强型绿色荧光蛋白（eGFP）的表达，从而获得了含有汉滩病毒糖蛋白G2的重组腺病毒。将重组腺病毒用于感染人宫颈癌（Hela）细胞，并通过间接免疫荧光法和蛋白质免疫印迹法检测表达的蛋白。在多个层面证实了构建体：首先，限制性内切酶分析表明重组腺病毒载体构建正确；其次，逆转录-聚合酶链反应（RT-PCR）显示G2基因能在Hela细胞中正确转录。然后，荧光显微镜证明了感染的Hela细胞中eGFP的表达。最后，间接免疫荧光法和蛋白质免疫印迹法证实了用抗G2单克隆抗体鉴定的目的蛋白的表达。总之，本研究成功构建了含有汉滩病毒包膜糖蛋白G2的重组腺病毒，同时获得了G2蛋白，这可能为G2蛋白的结构研究和汉滩病毒新型疫苗奠定坚实基础。
