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目的:构建带有绿色荧光蛋白(GFP)标签的汉滩病毒(HTNV)包膜糖蛋白(GP)真核表达载体,并进行初步表达鉴定。方法:以peGFP-C1质粒为模板扩增GFP片段,将其插入真核表达载体pFLAG-CMV3中,构建成可表达带有GFP标签分泌型蛋白的载体pFLAG-CMV3-GFP。扩增HTNV-G1、HTNV-G2,分别插入上述载体,转染293T细胞,采用检测GFP标签蛋白的夹心ELISA法检测转染细胞培养上清中分泌型重组蛋白的含量。结果:酶切及测序证实带有GFP标签的HTNV-G1、HTNV-G2表达载体构建成功,分别命名为pFLAG-G1-GFP/pFLAG-G2-GFP,转染细胞的培养上清中可以检测出带有GFP标签的融合蛋白。结论:成功构建了带有GFP标签HTNV-GP的重组质粒并成功表达,为深入研究HTNV感染后机体针对HTNV-GP的特异性免疫应答规律奠定了实验基础。
OBJECTIVE: To construct a eukaryotic expression vector of glycoprotein (GP) of Hantaan virus (HTNV) tagged with green fluorescent protein (GFP) and to identify its expression. Methods: GFP fragment was amplified from peGFP-C1 plasmid and inserted into eukaryotic expression vector pFLAG-CMV3 to construct pFLAG-CMV3-GFP, a vector expressing GFP-tagged secreted protein. The HTNV-G1 and HTNV-G2 were amplified and transfected into 293T cells respectively. The sandwich ELISA was used to detect the secreted recombinant protein in the supernatant of transfected cells. Results: After transfected with pFLAG-G1-GFP / pFLAG-G2-GFP, the HTNV-G1 and HTNV-G2 expression vectors with GFP tag were confirmed by restriction enzyme digestion and sequencing. GFP tagged fusion protein. CONCLUSION: The recombinant plasmid with GFP-tagged HTNV-GP was successfully constructed and successfully expressed, which lays the foundation for further study of the regulation of HTNV-GP specific immune response after HTNV infection.