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目的:构建针对端粒酶hTERT基因的U6/H1双启动子siRNA表达框架(SEC),并观察其转录产物对HeLa细胞端粒酶活性的干扰作用。方法:利用融合PCR技术,针对人端粒酶hTERT基因开放阅读区构建3条U6/H1双启动子SEC以及针对其3’端非翻译区构建1条U6/H1双启动子SEC,对各SEC鉴定后,分别转染人宫颈癌HeLa细胞,用端粒重复序列扩增法(TRAP)检测HeLa细胞的端粒酶活性。结果:4种针对端粒酶hTERT基因的U6/H1双启动子SEC均成功构建,转染HeLa细胞后,对端粒酶活性的抑制率分别为36.8%、57.39%、80.47%、70.31%。结论:针对端粒酶hTERT基因的U6/H1双启动子SEC的成功构建,为开展肿瘤端粒酶基因干扰的实验研究提供了新的有效手段。
OBJECTIVE: To construct a double-promoter U6 / H1 siRNA expression vector targeting telomerase hTERT gene and to observe its interference effect on telomerase activity in HeLa cells. METHODS: Three U6 / H1 double-promoter SECs were constructed based on the open reading frame of human telomerase hTERT gene by fusion PCR and one U6 / H1 double-promoter SEC was designed for the 3 ’untranslated region. After identification, HeLa cells were transfected with HeLa cells respectively, and the telomerase activity of HeLa cells was detected by telomeric repeat amplification (TRAP). Results: Four U6 / H1 double promoters against telomerase hTERT gene were successfully constructed. The inhibitory rates of telomerase activity were 36.8%, 57.39%, 80.47% and 70.31%, respectively. Conclusion: The successful construction of the U6 / H1 double promoter SEC targeting telomerase hTERT gene provides a new and effective method for the experimental study on the telomerase gene interference.