吉林大学学报(医学版) ›› 2019, Vol. 45 ›› Issue (05): 997-1002.doi: 10.13481/j.1671-587x.20190504

• 基础研究 • 上一篇    

miR-186过表达慢病毒载体的构建及鉴定

李胜男1, 王梦旭2, 胡伟东2, 陈少凤2, 陈杏兰2, 李友1   

  1. 1. 广东省衰老相关心脑疾病重点实验室, 广东 湛江 524002;
    2. 广东医科大学附属医院神经病学研究所, 广东 湛江 524002
  • 收稿日期:2018-11-23 发布日期:2019-10-08
  • 通讯作者: 李友,副研究员,硕士研究生导师(Tel:0759-2386772,E-mail:youli805@163.com) E-mail:youli805@163.com
  • 作者简介:李胜男(1992-),女,安徽省淮北市人,研究实习员,理学硕士,主要从事神经退行性疾病基础方面的研究。
  • 基金资助:
    国家自然科学基金资助课题(81571157)

Construction and identification of miR-186 overexpression lentiviral vector

LI Shengnan1, WANG Mengxu2, HU Weidong2, CHEN Shaofeng2, CHEN Xinglan2, LI You1   

  1. 1. Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Zhanjiang 524002, China;
    2. Institute of Neurology, Affiliated Hospital, Guangdong Medical University, Zhanjiang 524002, China
  • Received:2018-11-23 Published:2019-10-08

摘要: 目的:构建miR-186过表达慢病毒载体并包装慢病毒,探讨miR-186在人胚胎肾细胞(HEK)293T细胞系中的感染效率和表达水平。方法:以Hsa-miR-186前体序列为模板,设计并合成引物,PCR法扩增pre-miR-186基因序列,并将其克隆到携带EGFP/Puromycin的慢病毒载体FV040中,经EcoRⅠ和AgeⅠ酶切及测序鉴定后获得重组慢病毒载体。利用Lipofectamine 2000将重组慢病毒质粒FV040 Vector和FV040 miR-186分别与辅助质粒通过共转染至HEK293T细胞中,48 h后收集慢病毒,以FV040 Vector慢病毒作为对照组,FV040 miR-186作为实验组,分别感染HEK293T细胞。感染48 h后,观察HEK293T细胞中绿色荧光的分布情况,并采用实时荧光定量PCR法检测miR-186的表达水平。结果:测序分析,miR-186过表达慢病毒与GenBank上公布的miR-186序列完全一致。与对照组(0.8387±0.1456)比较,实验组HEK 293T细胞中miR-186表达水平(12.6400±0.7884)明显升高(t=14.72,P<0.01),约为对照组的15.07倍。结论:成功构建miR-186过表达慢病毒载体并包装出慢病毒,miR-186慢病毒成功感染HEK293T细胞,miR-186表达水平在HEK293T细胞中明显升高。

关键词: miR-186, 慢病毒, HEK293T细胞, 过表达慢病毒载体, 绿色荧光蛋白

Abstract: Objective:To construct the miR-186 overexpression lentiviral vector and package the lentivirus, and toexplore the infection efficiency and the expression level of miR-186 in the HEK293T cells. Methods:The Hsa-miR-186 precursor sequence was used as a template to design and synthesize the primer, and the the pre-miR-186 gene was amplified by PCR. The pre-miR-186 gene sequence was cloned into the lentiviral vector FV040 carrying EGFP/Puromycin cassette. The recombinant lentiviral vector was digested by EcoRⅠand AgeⅠrestriction endonuclease and confirmed by sequencing. The recombinant FV040 Vector and FV040 miR-186 were co-transfected into the HEK293T cells with the helper plasmids using Lipofectamine 2000, respectively; the FV040 Vector lentivirus (control group) and the FV040 miR-186 lentivirus (experiment group) were collected and used to infect the HEK293T cells 48 h after transfection, respectively. The green fluorescence distribution in the HEK 293T cells was observed 48 h after transfection, and the expression level of miR-186 was determined by real-time fluorescence quantitative PCR. Results:The sequencing analysis results indicated that the sequence of miR-186 overexpressing lentivirus was identical with the sequence of miR-186 published on GenBank. The titers in control group and experiment group were 6×108 TU·mL-1 and 5×108 TU·mL-1, respectively. The relative expression level of miR-186 in the HEK293T cells in experiment group (12.640 0±0.788 4) was significantly increased by 15.07 times (t=14.72,P<0.01) compared with control group (0.838 7±0.145 6). Conclusion:The lentiviral vector which overexpresses miR-186 is constructed successfully and the miR-186 lentivirus is prepared. The HEK 293T cells are infected with miR-186 lentivirus suceessfully and the expression level of miR-186 in the HEK 293T cells is increased significantly.

Key words: miR-186, lentivirus, HEK293T cells, overexpression lentiviral vector, green fluorescence protein

中图分类号: 

  • R346