摘要
目的克隆hTERT启动子核心序列,研究hTERT启动子/SV40增强子在食管癌细胞中的联合转录活性。方法以人基因组DNA为模板,PCR扩增hTERT启动子核心片段;将其分别插入荧光素酶基因报告质粒pGL3-Basic和pGL3-Enhancer中,构建hTERT启动子调控的表达载体pGL3-hTERTp和由hTERT启动子/SV40增强子联合调控的表达载体pGL3-hTERTp-SV40en,将上述重组质粒分别瞬时转染食管癌细胞Eca-109、EC1和人胚肺成纤维细胞MRC-5,用荧光素酶检测试剂盒检测转染细胞中荧光素酶基因的表达水平并以此计算hTERT启动子和hTERT启动子/SV40增强子在各种细胞中的转录活性。结果克隆出长213bp的hTETR启动子核心片段,DNA测序结果与GenBank中hTERT启动子的碱基序列完全一致;成功构建真核表达载体pGL3-hTER-Tp和pGL3-hTERTp-SV40en;hTETR启动子在食管癌细胞Eca-109和EC1中均有转录活性,在MRC-5细胞中无明显转录活性;hTERT启动子/SV40增强子在食管癌细胞Eca-109和EC1中的转录活性显著高于hTETR启动子的单独转录活性。结论 hTERT启动子在食管癌细胞中具有靶向性转录活性,SV40增强子能显著增强hTERT启动子在食管癌细胞中的转录活性,有可能作为肿瘤靶向性基因治疗的转录调控元件。
Objective To clone core sequence of hTERT promoter,and study transcriptional activity of hTERT promoter/SV40 enhancer in esophageal cancer cell.Methods hTERT promoter was amplified from human genomic DNA using polymerase chain reaction（PCR）;hTERT promoter was inserted into pGL3-Basic and pGL3-enhancer to construct luciferase gene expression driven by hTERT promoter（named as pGL3-hTERTp） or by hTERT promter/SV40 enhancer（named as pGL3-hTERTpSV40en）,respectively.The recombinants were transiently transfected into esophageal cancer cells of Eca-109,EC1 and human embryo lung fibroblast MRC-5,respectively,then expression level of luciferase gene in transfected cells was studied to evaluate transcriptional activities of hTERT promoter and hTERT promoter/SV40 enhancer in esophageal cancer cells.Results A 213bp core-sequence of hTETR promoter was cloned successfully,and DNA sequencing showed its sequence the same as that registered in GenBank;recombinants of pGL3-hTERTp and pGL3-hTERTp-SV40en were successfully constructed.hTERT promoter had transcriptional activity in esophageal cancer cells while no transcriptional activity in MRC-5 cells;The transcriptional level of hTERT promoter/SV40 enhancer in esophageal cancer cells was significantly higher than hTERT promoter alone.Conclusion hTERT promoter has selective transcriptional activity in esophageal cancer cells;SV40 enhancer could significantly elevate transcriptional level of hTERT promoter,and it could play an important role in targeting gene therapy for tumor.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2010年第11期1230-1233,共4页
Cancer Research on Prevention and Treatment
