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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 14-3-3σ interference retroviral vector and establish stable transfected HaCat cell line. Methods: The interfering sequence of 14-3-3σ gene was synthesized and inserted into pSuper-retro-neo-EGFP plasmid. The plasmid was amplified in STBL3 and positive clones were selected by brushing. The positive clones were identified by restriction enzyme digestion and transfected into 293FT cells The virus was packaged, amplified, and purified. The retrovirus vector was obtained. The HaCat cells were infected with retroviral vector and Western-blotting was used to detect the expression of 14-3-3σ by Real-time PCR. Results: The recombinant plasmid pSuper-retro-neo-EGFP-si14-3-3σ was cloned by restriction enzyme digestion and sequencing. The HaCat cells stably transfected with interference plasmids showed green fluorescence under inverted fluorescence microscope, western blot and Real -timePCR method showed that 14-3-3σ expression was significantly inhibited. Conclusion: The 14-3-3σ interference retroviral vector was successfully constructed and its stable transfected HaCat cell line was constructed.