miR-137过表达慢病毒的包装和鉴定

发布时间：2018-03-20 19:46

本文选题：miR-　切入点：慢病毒　出处：《吉林大学学报(医学版)》2017年04期 　论文类型：期刊论文

【摘要】：目的:构建miR-137过表达慢病毒载体并进一步包装慢病毒,探讨miR-137在HEK293T细胞中的感染效率和表达水平。方法:化学合成miR-137序列并克隆到慢病毒载体GV209,得到并鉴定含有目的片段的重组质粒,采用Lipofectamine 2000共转miR-137重组质粒与慢病毒辅助包装质粒到HEK293T细胞中生产慢病毒。以感染复数(MOI)值为40感染HEK293T细胞48h后,荧光显微镜下观察细胞中绿色荧光蛋白(GFP)的表达,采用荧光定量PCR法检测HEK293T细胞中miR-137的表达水平。结果:测序结果,目的基因序列与GenBank公布的miR-137基因序列完全一致。在感染的HEK293T细胞中观察到GFP的表达。过表达慢病毒感染细胞中miR-137表达水平是对照细胞的12.74倍。结论:成功包装miR-137过表达慢病毒,并可高效感染HEK293T细胞。
[Abstract]:Objective: to construct miR-137 overexpression lentivirus vector and further package lentivirus. Methods: the miR-137 sequence was chemically synthesized and cloned into lentivirus vector GV209, and the recombinant plasmid containing the target fragment was obtained and identified. Lentivirus was produced by co-transfer of Lipofectamine 2000 miR-137 recombinant plasmid and lentivirus assisted packaging plasmid into HEK293T cells. The expression of green fluorescent protein (GFP) in HEK293T cells was observed by fluorescence microscope after 48 h infection of HEK293T cells with a number of infected plural moi values of 40. Fluorescence quantitative PCR assay was used to detect the expression of miR-137 in HEK293T cells. Objective the gene sequence was completely consistent with the miR-137 gene sequence published by GenBank. The expression of GFP was observed in infected HEK293T cells. The expression level of miR-137 in lentivirus infected cells was 12.74 times higher than that in control cells. And it can infect HEK293T cells efficiently.
【作者单位】：广东医科大学附属医院精神心理科;广东医科大学神经病学研究所;
【基金】：国家自然科学基金资助课题(81670252) 广东省科技厅自然科学基金资助课题(2015A030313523) 广东省湛江市科技局科技计划项目资助课题(2016A01008)
【分类号】：R749.3

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