天津医药

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p100蛋白慢病毒干扰载体及p100沉默的HepG2稳定细胞系的构建

黄丽1,尹洁2,丁建民3,宋娟1,支蕾4,何津岩1,张毅1,高星杰2,经翔3,杨洁1   

  1. 1. 天津医科大学免疫教研室
    2. 天津医科大学基础医学研究中心
    3. 天津市第三中心医院超声科
    4. 天津医科大学免疫学教研室
  • 收稿日期:2011-11-28 修回日期:2012-02-09 出版日期:2012-06-15 发布日期:2012-06-15
  • 通讯作者: 黄丽

Construction of a p100 Stable Knock-downed HepG2 Cell Line with a p100 shRNA Expression Lentiviral System

  • Received:2011-11-28 Revised:2012-02-09 Published:2012-06-15 Online:2012-06-15

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摘要： 目的：用表达shRNA的干扰慢病毒载体系统建立p100基因稳定沉默的HepG2人肝癌细胞系。方法：通过RNAi序列设计软件进行p100干扰片段设计，筛选出p100基因的RNAi有效靶序列，进一步合成靶序列的Oligo DNA并构建pLKO.3G-p100I慢病毒载体，用双酶切及测序进行鉴定。将构建好的p100 shRNA表达质粒与慢病毒包装质粒共转染至包装细胞293T，包装产生病毒颗粒。运用病毒颗粒感染HepG2细胞，获得p100稳定沉默的细胞系。荧光显微镜下观察感染效率，利用Western blot方法检测稳定细胞株中p100的沉默效果。结果：成功构建了具有p100干扰效果的慢病毒载体，并获得了稳定沉默p100及对照的HepG2细胞株。慢病毒感染HepG2细胞后，荧光显微镜下观察到90%以上细胞发出绿色荧光，Western blot实验证实干扰p100后，HepG2细胞株中p100蛋白表达水平显著降低。结论：成功构建了具有p100干扰效果的慢病毒干扰载体及p100稳定沉默的HepG2细胞系。

关键词: RNA干扰, 细胞系, 肿瘤, 质粒, 慢病毒载体, p100蛋白, HepG2细胞系

Abstract: Objective: To construct a lentiviral vector expressing a short hairpin RNA (shRNA) which targets the functional p100 gene and to construct a p100 stable knock-downed HepG2 cell line with this p100 shRNA expressing lentiviral system. Methods: A double-stranded shRNA targeting the p100 gene was designed, synthesized and cloned into the pLKO.3G vector. Positive clones were verified with double enzyme digestion and sequencing. Then the valid constructions were cotransfected with the other 2 packaging plasmids into 293T cells to package lentiviral particles carrying p100 shRNA expression element. The lentivirus was then harvested and used to infect HepG2 cells. Infection efficiency was indicated with GFP and detected with fluorescent microscope. The knock-down efficiency of p100 in infected HepG2 cells was verified with western blot. Results: A lentiviral vector carrying a shRNA targeting the p100 gene was successfully constructed and a p100 stable knock-downed HepG2 cell line was established by using this lentiviral system. After viral particles infection of HepG2 cells, strong green fluorescence was observed in HepG2 cells under fluorescent microscope, the infection efficiency exceeded 90%. Western blot analyses confirmed that the expression of p100 was significantly down-regulated in this infected HepG2 cell line. Conclusion: A lentiviral vector carrying an shRNA targeting the p100 gene was successfully constructed and a HepG2 cell line stably expressing p100 shRNA was established with this lentiviral system.

Key words: RNA interference, cell line, tumor, plasmids, lentiviral vector, p100 protein, HepG2 cell line