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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。
The aim of this study was to construct a recombinant adenovirus expressing Hantaan virus (HTNV) glycoprotein G2. The G2 gene was amplified by PCR and cloned into T / A cDNA. The recombinant plasmid was subcloned into the adenovirus shuttle vector pAd5-CMV and the recombinant adenovirus shuttle vector carrying the G2 gene was cloned into the adenovirus shuttle vector Recombinant adenovirus carrying HTNV glycoprotein G2 gene was packaged, amplified, and purified. The recombinant adenovirus was transfected into HEK293 cells to infect Hela cells for protein harvesting, indirect immunofluorescence and Western blotting Protein expression was detected. Restriction enzyme digestion indicated that recombinant adenovirus vector carrying G2 gene was constructed successfully. RT-PCR analysis indicated that the target gene could be transcribed in Hela cells infected with recombinant adenovirus. Hela cells infected with recombinant adenovirus were observed by fluorescence microscopy. The expression of eGFP was confirmed by indirect immunofluorescence assay and Western blotting. The product was confirmed by anti-G2 monoclonal antibody, indicating that glycoprotein G2 was expressed in infected cells. The recombinant adenovirus expressing HTNV envelope glycoprotein G2 was successfully constructed in this study. The transfection of host cells can stably express the target protein, which lays the foundation for the crystal structure analysis of HTNV glycoprotein G2 and the development of a novel Hantavirus vaccine .