Construction of Cytosine Deaminase:Uracil Phosphoribosyltransferase/5-fluorocytosine Gene Therapy System under Control of Human Telomerase Reverse Transcriptase Promoter and Its Application

:Objective 　To const ruct the expression vector containing CD :UPRT(cytosine deaminase :uracil phosphoribosylt ransferase) genes under the cont rol of the hTERT promoter and investigate it s specific killing effect s on human gast ric cancer cells SGC7901 in vi t ro. Methods 　The hTERT promoter was PCR amplified and cloned into the p GL32Basic vector. The recombinant was t ransfected into SGC7901 cells and normal human fibroblast cells HL F to detect the t ranscriptional activities of the hTERT gene promot2 er. The expression vector containing CD :UPRT genes under the cont rol of the hTERT promoter named as hTERT2CD :UPRT was const ructed. This vector and the vector containing CD :UPRT genes under the cont rol of cytomegalovirus ( CMV) promoter named as pcDNA3. 12CD : UPRT were t ransfected into SGC7901 and HL F cells, respectively. The t ransfected cells were selected by G418. The expression of the CD gene was detected by RT2PCR and Western blot . MTT analysis was used to determine the cyto2 toxic effect s of the CD :UPRT/ 52FC system. Results 　The hTERT promoter was PCR amplified success2 fully. Luciferase assay showed the relative luciferase activity of SGC7901 by the hTERT promoter was (21. 50 ±2. 15) % and that of HL F cells was only (0. 40 ±0. 07) %. The expression vector hTERT2CD : UPRT was successfully const ructed. Af ter stably t ransfected with pcDNA3. 12CD :UPRT, SGC7901 and HL F cells both expressed CD genes and were sensitive to 52FC, while positive only in SGC7901 cells af2 ter stably t ransfected with pcDNA 3. 12CD :UPRT. Conclusion 　The hTERT promoter can specifically cont rol the CD :UPRT gene expression in SGC7901 cells but not in the normal cells and the CD :UPRT/52FC system under cont rol of the hTERT promoter can specially kill SGC7901 cells in vi t ro.