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

作　　者：张璐玢 谢梦晓 陈云鹏[1] 徐晓昱[1] 张彩云[1] 冯晖晖 刘霞[1] 周小明[1] 夏圣[1] 邵启祥[1] ZHANG Lu-bin XIE Meng-xiao CHEN Yun-peng XU Xiao-yu ZHANG Cai-yun FENG Hui-hui LIU-xia ZHOU Xiao-ming XIA-sheng SHAO Qi-xiang(School of Medicine, Jiangsu Unlversity, Zhenjiang Jiangsu 212013, China)

机构地区：[1]江苏大学医学院,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2016年第1期26-30,共5页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(31400773);江苏省高校自然科学基金资助项目(14KJB320001);江苏省高等学校大学生创新创业训练计划项目(3121502020)

摘　　要：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.

关 键 词：microRNA-31 反转录病毒载体 重组质粒 

分 类 号：R393[医药卫生—基础医学]