端粒酶逆转录酶基因启动子调控的肿瘤细胞特异表达载体的构建和鉴定

Construction and identification of a tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor

目的 利用端粒酶逆转录酶基因启动子 ,构建能在人肿瘤细胞中特异表达目的基因的表达载体。方法 通过PCR方法从 pEGFP N1质粒中扩增出绿色荧光蛋白基因并克隆到逆转录病毒载体pLNCX的多克隆位点上 ,命名为 pLNCX EGFP。设计含有特定酶切位点的引物 ,从人基因组中扩增出端粒酶逆转录酶基因启动子序列 ,将该序列片段定向克隆到已用限制性内切酶切除巨细胞病毒启动子的 pLNCX EGFP载体上 ,构建成端粒酶逆转录酶基因启动子调控的含有绿色荧光蛋白报告基因的表达载体 pLNT EGFP。将该表达载体pLNT EGFP瞬时转染高表达端粒酶活性的HLF细胞株和不表达端粒酶活性的WI38细胞株 ,以检测端粒酶逆转录酶基因启动子的转录活性。结果 表达载体pLNT EGFP经酶切、测序分析证实与设计的完全一致。细胞瞬时转染结果表明 ,pLNT EGFP能特异地在端粒酶阳性细胞中高效表达绿色荧光蛋白报告基因 ,而在端粒酶阴性细胞中不表达报告基因。结论 成功构建了由端粒酶逆转录酶基因启动子调控的肿瘤细胞靶向表达载体。

Objective To construct the tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor. Methods The fragment of the enhanced green fluorescent protein (EGFP) gene was PCR amplified from the pEGFP-N1 plasmid and cloned into the multiple cloning site of pLNCX vector, and the recombinant was named as pLNCX-EGFP. The fragment of human telomerase reserve transcriptase gene promoter was amplified from the human genome by using the human telomerase reserve transcriptase gene specific primers, and cloned into the pLNCX-EGFP vector, where the cytomegalovirus promoter was previously removed using restriction enzymes, in sense orientation relative to the green fluorescent protein coding sequence. Then the expression vector pLNT-EGFP under the control of the human telomerase reserve transcriptase gene promoter, containing green fluorescent protein reporter gene, was successfully constructed. To detect the transcriptional activity of the human telomerase reserve transcriptase gene promoter, transient transfection of this specific expression vector into HLF cell lines with high telomerase activity and WI38 cell lines without telomerase activity was performed. Results The expression vector proven by restriction enzymes digestion and sequencing was in correspondence with the design. The results of transient transfection showed that the pLNT-EGFP vector could highly expressed green fluorescent protein reporter gene in telomerase-positive cells, but not in telomerase-negative cells. Conclusion A tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor has been successfully constructed.