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目的:以p MSCV-puro载体(MGP)为骨架,构建带有绿色荧光蛋白(green fluorescent protein,GFP)的小鼠micro RNA-31(mi R-31)反转录病毒载体MGP-31。方法:PCR扩增带有NotⅠ以及XhoⅠ酶切位点的mi R31的前体(premi R-31)序列,然后克隆至MGP载体上,测序鉴定。将构建的MGP-31质粒转染HEK-293T细胞,通过荧光显微镜观察GFP的表达情况,实时定量PCR检测mi R-31转录水平以评估MGP-31质粒的转染效率。结果:基因序列分析显示pre-mi R-31基因序列正确,并成功插入MGP载体,转染HEK-293T细胞可表达GFP,实时定量PCR结果表明转染MGP-31质粒的HEK-293T细胞可高表达mi R-31。结论:MGP-31反转录病毒载体构建成功。
OBJECTIVE: To construct the mouse micro RNA-31 (mi R-31) retroviral vector MGP-31 with green fluorescent protein (GFP) using pGPV-puro vector (MGP) as the backbone. Methods: The mi R31 precursor (premi R-31) sequence with Not Ⅰ and Xho Ⅰ restriction sites was amplified by PCR, then cloned into MGP vector and sequenced. The constructed MGP-31 plasmid was transfected into HEK-293T cells, the expression of GFP was observed by fluorescence microscope, and the mi R-31 transcription level was detected by real-time quantitative PCR to evaluate the transfection efficiency of MGP-31 plasmid. Results: The gene sequence analysis showed that the pre-mi R-31 gene sequence was correctly inserted into MGP vector and transfected into HEK-293T cells to express GFP. Real-time quantitative PCR results showed that HEK-293T cells transfected with MGP-31 plasmid were high Express mi R-31. Conclusion: MGP-31 retroviral vector was constructed successfully.