miR-137过表达慢病毒的包装和鉴定

doi:10.13481/j.1671-587x.20170406

吉林大学学报(医学版) ›› 2017, Vol. 43 ›› Issue (04): 694-697.doi: 10.13481/j.1671-587x.20170406

miR-137过表达慢病毒的包装和鉴定

律东¹, 梁春梅², 李明颖¹, 殷静雯¹, 罗旭东¹, 林举达¹, 马国达²

1. 广东医科大学附属医院精神心理科, 广东湛江 524001;
2. 广东医科大学神经病学研究所, 广东湛江 524001

收稿日期:2017-03-01 出版日期:2017-07-28 发布日期:2017-08-01
通讯作者: 马国达,副教授,研究生导师(Tel:0759-2386772,E-mail:sihan1107@126.com) E-mail:sihan1107@126.com
作者简介:律东(1979-),男,黑龙江省哈尔滨人,主治医师,主要从事精神分裂症基础和临床方面的研究。
基金资助:
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）

Packaging and identification of miR-137 overexpression lentivirus

LYU Dong¹, LIANG Chunmei², LI Mingying¹, YIN Jingwen¹, LUO Xudong¹, LIN Juda¹, MA Guoda²

1. Department of Psychiatry, Affiliated Hospital, Guangdong Medical University, Zhanjiang 524001, China;
2. Institute of Neurology, Guangdong Medical College, Zhanjiang 524001, China

Received:2017-03-01 Online:2017-07-28 Published:2017-08-01

摘要/Abstract

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。

关键词: HEK293T细胞, miR-137, 慢病毒, 绿色荧光蛋白

Abstract: Objective: To construct lentiviral vector which can overexpression miR-137and produce lentivirus by lentivirus packaging system, and to explore its infection efficiency and expression in HEK293T cells.Methods: miR-137 sequence was chemically synthesized and cloned into lentiviral vector GV209, and the recombinant plasmid containing human miR-137 was obtained and identified. Then miR-137 recombinant plasmid together with two helper plasmids were transfected into HEK293T cells using Lipofectamine 2000.After the HEK293T cells were infected in multiplicity of infection(MOI) 40 for 48 h, the expression of green fluorescent protein (GFP) was observed by fluorescence microscope and the expression level of miR-137 was detected by fluorescence quantitative PCR.Results: The sequencing results showed that the inserted gene sequence was completely consistent with the published human miR-137 gene sequence in GenBank. The GFP was observed in the HEK293T cells infected with miR-137 overexpression lentivirus under fluorescence microscope. The fluorescence quantitative PCR results showed that the expression level of miR-137 in the cells infected with overexpression lentivirus was 12.74 times higher than that in the control cells.Conclusion: The lentivirus containing miR-137 gene is successful packaged, and it could efficiently infect the HEK293T cells.

Key words: miR-137, lentivirus, HEK293T cell, green fluorescent protein

中图分类号:

Q786

律东, 梁春梅, 李明颖, 殷静雯, 罗旭东, 林举达, 马国达. miR-137过表达慢病毒的包装和鉴定[J]. 吉林大学学报(医学版), 2017, 43(04): 694-697.

LYU Dong, LIANG Chunmei, LI Mingying, YIN Jingwen, LUO Xudong, LIN Juda, MA Guoda. Packaging and identification of miR-137 overexpression lentivirus[J]. Journal of Jilin University Medicine Edition, 2017, 43(04): 694-697.

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miR-137过表达慢病毒的包装和鉴定

Packaging and identification of miR-137 overexpression lentivirus

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