hTERT启动子核心序列的克隆及其在肺癌细胞中靶向转录活性的研究

A Study on Cloning Core-sequence of hTERT Promoter and Its Targeting Transcriptional Activity in Lung Cancer Cells

背景与目的构建人端粒酶催化亚单位（hTERT）启动子核心片段调控的绿色荧光蛋白（EGFP）基因表达质粒，并初步研究hTERT启动子在肺癌细胞和正常细胞中的转录活性。 方法以人胚肾293细胞（HEK293）基因组DNA为模板，应用PCR方法，克隆hTERT启动子核心片段，将其插入无启动子的绿色荧光蛋白（EGFP）基因报告质粒载体pEGFP—1的多克隆位点中，构建成hTETR启动子调控的EGFP表达质粒pEGFP—1—hTERTp；用脂质体转染法将pEGFP—1—hTERTp分别转染肺癌细胞A549和人胚肺成纤维细胞MRC-5，观察hTERT启动子在上述细胞中的转录活性。

Background and objective To construct green fluorescence protein gene expression plasmid driven by core-sequence of hTERT promoter and originally explore transcriptional character of hTERT promoter in human lung cancer cells and normal cells. Methods 1.A 238 bp core-sequence of the hTERT promoter was amplified from genomic DNA isolated from 293 cells by polymerase chain reaction （PCR）; 2. Green fluorescent protein（EGFP） gene expression plasmid named pEGFP-1- hTERTp was constructed by insertion of hTERT promoter into upstream area of EGFP gene in plasmid of pEGFP-l,which has no promoter; 3. The recombinant of pEGFP-I-hTERTp was transiently transfected into human lung cancer cell A549 and human embryonic lung fibroblast MRC-5 respectively with lipofectamine method, and then expression of EGFP expression was observed in these cells to confirm transcriptional character ofhTERT promoter.