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目的观察不同长度的人端粒酶催化亚单位(hTERT)的启动子对启动增强型绿色荧光蛋白(EGFP)能力的差异,评价启动子启动功能与长度的关系。方法克隆4段不同长度的端粒酶基因起始位点(ATG)上游序列长约2 068、1 135、333和142 bp的启动子片段,通过观察EGFP的荧光强度,确定启动效率。结果显微镜观察荧光亮度及荧光分光光度计检测荧光强度随着启动子长度的缩短而减弱,荧光强度由高至低依次为8.56,7.81,6.88,3.62。结论人端粒酶的表达强度及端粒酶的活性变化与其启动子的长度有关,而肿瘤细胞的端粒酶表达相对增强,可通过下调启动子活性,为肿瘤的靶向性基因治疗提供新思路。
OBJECTIVE: To observe the differences in promoter capacity of human telomerase catalytic subunit (hTERT) promoter to enhanced green fluorescent protein (EGFP) and evaluate the relationship between promoter function and length. METHODS: Four promoter fragments of 2 068, 1 135, 333 and 142 bp upstream of the telomerase gene start site (ATG) of different lengths were cloned. The activation efficiency of EGFP was determined by observing the fluorescence intensity of EGFP. Results The fluorescence intensity was observed microscopically and the fluorescence intensity decreased with the decrease of the length of the promoter. The highest and lowest fluorescence intensities were 8.56, 7.81, 6.88 and 3.62, respectively. Conclusions The expression of human telomerase and telomerase activity are related to the length of its promoter, while the expression of telomerase in tumor cells is relatively enhanced. It can provide new target gene therapy for tumor by down-regulating the promoter activity Ideas.