Construction Of Luciferase Transcriptional Targeting Vectors Driven By HTERT Core Promoter Modified By SV40 Enhancer

ObjectiveAmplifying the human telomerase reverse transcriptase (hTERT) core promoter sequence by PCR and cloning it into luciferase reporter plasmids pGL3Basic and pGL3Enhancer which contains a SV40 enhancer in the polyA downstream to study the transcriptional activities of hTERT promoter and hTERT-SV40 chimeric promoter system in various telomerase positive cancercells and telomerase negative normal cell. We try to enhance the hTERT promoter specific transcriptional activity in tumor cell by SV40 enhancer and make a basis research of theory and experiment on gene therapy about tumor.MethodsThe hTERT promoter of 260bp was amplified with polymerase chain reaction (PCR) method, utilizing DNA of HT-29 cell as a template. After DNAsequencing with correct result, the hTERT core promoter was inserted into luciferase reporter vectors (pGL3Basic and pGL3Enhancer) to reconstruct a recombinant plasmids named pGL3hTP and pGL3hTP-SV40 . Then thepGL3hTP, pGL3hTP-SV40 pGL3-Control and pGL3-Basic separately with pRL-TK were transiently transfected into cancer cell lines HT-29, SW-620,MGC-803 and human fibroblast cell line MRC5. Among them pGL3-Basic takenas negative control? pGL3-Control taken as positive control and pRL-TK. taken as inner reference control. The transcriptional activities of hTERT promoter and hTERT-SV40 promoter system in various cells were determined by measuring theluciferase activities after 48 hours of transfection . The value ofpGL3-control/pRL-TK is taken as 100% and the other values are relative percent of it.ResultsElectrophoresis demonstrated that cloned hTERT promoter was about 26Obp, and DNA sequencing showed a same sequence as registered in GenBank. The cloned hTERT promoter was located between -204bp and +56bp in upstream of the transcription start site and the length was 260bp. The recombinant plasmids pGL3hTP and pGL3hTP-SV40 were confirmed by double restricted enzemy digestion and PCR method with correct results. The activity of different promoters(CMV\ hTP ^ hTP-SV40, )were assessed in tumor cell lines and one normal cell line by luciferase assays. The hTERT promoter activity was significantly higher in tumor cells than in normal cell(p>0.05). However, the hTERT-SV40 promoter activity was about 2-3 fold higher than the hTERT promoter in tumors cell lines and remained very weakly active in normal cell line.