小鼠microRNA-31反转录病毒载体重组质粒的构建及鉴定

Construction and identification of a microRNA-31 expression retroviral vector

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 a microRNA-31 expression retroviral vector （miR-31） containing green fluorescent protein （GFP） based on pMSCV-puro-vector. Methods： Murine miR-31 precursor sequence was amplified by PCR and inserted into MGP vector between the Not Ⅰ and Xho Ⅰ sites and named as MGP-31, the MGP-31 plasmid was identified by DNA sequencing. HEK-293T cells, transfected with the MGP-31 vec- tor, were verified with a fluorescence microscope to detect the expression of GFP. Meanwhile, miR-31 tran- scriptional level was detected by real-time PCR. Results： The result of sequencing showed that the sequence of pre-miR-31 was correct and inserted into the MGP vector successfully. GFP was detectable in HEK-293T cells transfected with MGP-31 vector. The result of real-time PCR showed that the miR-31 was over-expressed in HEK-293T cells. Conclusion： The retroviral vector MGP-31 was constructed successfully.