Yin Jia-Jun, Hu Xiang, Fu Pan-feng, Yuan Xiao-dong, Zhang Jin-yu
Department of General Surgery, The 4th Affiliated Hospital of Dalian Medical University, Dalian 116001, China.
Zhonghua Yi Xue Za Zhi. 2003 Jul 25;83(14):1274-7.
To construct the tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor.
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.
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.
A tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor has been successfully constructed.
构建由人端粒酶逆转录酶基因启动子驱动的肿瘤特异性表达载体。
从pEGFP-N1质粒中PCR扩增增强绿色荧光蛋白（EGFP）基因片段，并克隆到pLNCX载体的多克隆位点，重组体命名为pLNCX-EGFP。使用人端粒酶逆转录酶基因特异性引物从人类基因组中扩增人端粒酶逆转录酶基因启动子片段，并克隆到pLNCX-EGFP载体中，其中先前使用限制性内切酶去除了巨细胞病毒启动子，相对于绿色荧光蛋白编码序列以正义方向插入。然后成功构建了在人端粒酶逆转录酶基因启动子控制下、含有绿色荧光蛋白报告基因的表达载体pLNT-EGFP。为检测人端粒酶逆转录酶基因启动子的转录活性，将该特异性表达载体瞬时转染到具有高端粒酶活性的HLF细胞系和无端粒酶活性的WI38细胞系中。
经限制性内切酶消化和测序验证的表达载体与设计相符。瞬时转染结果表明，pLNT-EGFP载体可在端粒酶阳性细胞中高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。
成功构建了由人端粒酶逆转录酶基因启动子驱动的肿瘤特异性表达载体。
