鼠VEGF红色荧光慢病毒载体的构建及其在293T细胞中的表达

Construction of Lentiviral Vector pTK208-mVEGF-IRES-DsRed Contains DsRed and mVEGF and Its Expression in 293T Cells

目的构建含鼠血管内皮细胞生长因子(mVEGF)红色荧光慢病毒载体,鉴定其在293T细胞中的表达。方法构建含mVEGF和红色荧光蛋白(DsRed)基因双顺反子重组慢病毒质粒;采用脂染法将慢病毒系统三质粒共转染293T细胞,转染后24 h和48 h荧光显微镜下观察DsRed表达,收集48 h和72 h病毒上清并感染靶细胞239T,荧光显微镜下观察DsRed表达情况,并行Western blot分析培养上清和胞浆内mVEGF表达。结果成功构建慢病毒表达质粒pTK208-mVEGF-IRES-DsRed,重组慢病毒滴度达5×106PFU/mL,并获得蛋白DsRed和mVEGF的表达。结论含mVEGF红色荧光慢病毒质粒成功介导mVEGF蛋白的表达,该载体有望为研究VEGF的病理生理学机制及基因靶向治疗提供有效的工具。

Objective To construct red fluorescent protein（DsRed） and mouse vascular endothelial growth factor（mVEGF） lentiviral expression vector in order to obtain the DsRed and VEGF expression in 293T cells.Methods The mVEGF was amplified from the lungs of fetal mouse by RT-PCR.The IRES-DsRed was obtained from plasmid pLV-tTRKRAB-red which contains IRES-DsRed sequence by PCR.Then they were cloned into lentiviral expression vector pTK-208.Human embryonic kidney 293T cells were co-transfected with the three plasmids by lipofectamine,and the expression of DsRed was examined under fluorescent microscope 24 h and 48 h after transfection.48 h and 72 h later the viral supernatant was used to infect 293T cells,the expression of DsRed was examined under fluorescent microscope and Western blot assays was used to examine the expression of mVEGF in cells and the culture medium.Results The recombinant lentiviral vector pTK208-mVEGF-IRES-DsRed was constructed,the virus titres were above 10^6 PFU/mL in the supernatant.After infection,DsRed and mVEGF proteins were successfully expressed in 293T cells.Conclusion The lentiviral vector pTK208-mVEGF-IRES-DsRed contains DsRed and mVEGF were successfully constructed and expressed in 293T cells,it could provide a basis for further researches on the pathophysiological mechanism of VEGF and lay the foundation of gene therapy.