人端粒酶催化亚基基因启动子调控的表达载体的构建

Construction of an expression vector under the control of hTERT promoter

目的:构建人端粒酶催化亚基(humantelomerase reversetranscriptase,hTERT)基因的核心启动子调控的荧光素酶报告载体,检测其在肿瘤细胞及正常细胞中表达的差异。方法:提取人HeLa细胞的基因组,利用PCR技术克隆hTERT基因的核心启动子。将其插入荧光素酶报告载体中,经SacI和HindIII酶切和PCR鉴定后,转染肿瘤细胞及正常细胞,观察两种细胞中荧光素酶基因表达的差异。结果:hTERT基因的启动子调控的表达载体在肿瘤细胞中呈高效表达;而在正常细胞中的表达极低。结论:hTERT基因的启动子具有肿瘤特异性,有可能解决肿瘤基因治疗中的靶向性问题。

AIM: To construct a luciferase expression vector driven by hTERT(human telomerase reverse transcriptase) core promoter and identify the transcriptional activity of the vector in tumor cells and normal cells. METHODS: hTERT gene core promoter was amplified by PCR (using) the total genomic DNA from HeLa cells as template. The amplified gene fragment was subsequently cloned into PGL3-basic vector. Then the expression vector was confirmed by restriction enzyme digestion and PCR analysis. The luciferase activity driven by the hTERT gene core promoter was identified after transient transfection of the (expression) vector into tumor cells and normal cells. RESULTS: The (luciferase) activity of the transfected tumor cells was high, and very low in transfected normal cells. CONCLUSION: hTERT gene core promoter is tumor-specific and may be useful in gene therapy of tumor.