| 周美娟丁振华.14-3-3σ 干扰逆转录病毒载体的构建及其稳定转染HaCat 细胞系的建立[J].,2011,11(9):1601-1604 |
| 14-3-3σ 干扰逆转录病毒载体的构建及其稳定转染HaCat 细胞系的建立 |
| Construction of RNAi Recombinant Retroviral Vector of 14-3-3P and ItsStably Transfected HaCat Cell Lines |
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| DOI： |
| 中文关键词: 14-3-3σ 逆转录病毒 稳定转染的HaCat 细胞系 |
| 英文关键词: 14-3-3σ retroviral vector stably transfected HaCat cell lines |
| 基金项目:国家自然科学基金项目（30970673）；广东省自然科学基金项目（9151022501000013）；
南方医科大学公共卫生与热带医学学院院长基金（GW200826） |
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| 中文摘要: |
| 目的：构建14-3-3σ 干扰逆转录病毒载体，建立稳定转染的HaCat 细胞系。方法：人工合成14-3-3σ 基因干扰序列并定向插
入到pSuper-retro-neo-EGFP 质粒，并在STBL3 菌内进行质粒扩增，刷选阳性克隆，酶切测序鉴定，转染293FT 细胞进行病毒包
装、扩增、纯化、获取逆转录病毒载体，将逆转录病毒载体感染HaCat 细胞后Western 免疫印迹法、Real-time PCR 法检测14-3-3σ
的表达情况。结果：连接重组后经酶切和测序筛选出pSuper-retro-neo-EGFP-si14-3-3σ；干扰质粒稳定转染的HaCat 细胞系在倒置
荧光显微镜下呈绿色荧光，Western 免疫印迹法和Real-time PCR 法表明14-3-3σ 表达明显抑制。结论：成功构建了14-3-3σ 干扰
的逆转录病毒载体，并构建了其稳定转染的HaCat 细胞系。 |
| 英文摘要: |
| Objective: To construct the RNAi retroviral vector of 14-3-3σ and establish the stable transfected HaCat cell lines.
Methods: Hairpin siRNA of 14-3-3σ was synthesized and inserted into pSuper-retro-neo-EGFP plasmid. PSuper-retro-neo-
EGFP-si14-3-3σ was transformed into competent STBL3 cells. Then the positive clones were confirmed by sequencing and transfected
into the packaging 293FT cells to amplificate and depurate virus. HaCat cells were infected by the recombinant retroviral vector and the
expression of 14-3-3σ was detected by Western blot and real time PCR. Results: The recombinant retroviral plasmid PSuperretro-
neo-EGFP-si14-3-3σ was successfully constructed and green fluorescence of the stable transfected HaCat cell lines were observed
under inverted fluorescence microscope. The expression of 14-3-3 was down-regulated by the RNAi-14-3-3σ. Conclusion: The RNAi
retroviral vector targeting 14-3-3σ was successfully constructed and stably transfected HaCat cell lines were established. |
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