摘要
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.
出处
《中国肺癌杂志》
CAS
2009年第6期466-467,共2页
Chinese Journal of Lung Cancer