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 Its Stably Transfected HaCat Cell Lines

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-timePCR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-timePCR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。

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-neoEGFP-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 PSuper- retro-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.