前列腺特异性膜抗原启动子增强子调控UPRT基因真核质粒的构建及表达  

Construction and Expression of Recombinant Plasmid with Prostate-specific Membrane Antigen Promoter and Enhancer Regulating Uracil Phosphoribosyltransferase Gene

作　　者：赵福军[1] 李虹[1] 程鸿鸣[1] 曾浩[1] 魏强[1] 李响[1] 

机构地区：[1]四川大学华西医院泌尿外科,成都610041

出　　处：《四川大学学报（医学版）》2005年第2期172-175,共4页Journal of Sichuan University(Medical Sciences)

基　　金：四川省科技厅基金 (课题编号 0 4JY0 2 9-0 82 -1)资助

摘　　要：目的　构建前列腺特异性膜抗原启动子增强子靶向性调控的 U PRT基因真核表达重组质粒 (p PS-MAenhancer/ promoter- U PRT)。方法　采用 PCR技术从大肠杆菌 JM10 9基因组中扩增 U PRT基因 ,通过分子克隆技术将其克隆到包括前列腺特异性膜抗原启动子增强子靶向性调控的 p EGFP- 1的质粒。利用重组质粒 p PS-MAenhancer/ promoter- U PRT)转染 L Ncap细胞 ,MTT法检测 5 -氯尿嘧啶 (5 - FU)对转染 L Ncap细胞存活率的影响。结果　质粒 p PSMAenhancer/ promoter- EGFP双酶切去除 EGFP,连接上 U PRT基因 ,成功构建 p PSMAenhancer/ promoter- UPRT,并转染 L Ncap细胞 ,使 L Ncap细胞对 5 - FU的杀伤敏感性大大提高。结论　 p PSMAenhancer/ promoter- U PRT的构建和表达 ,为进一步研究 UPRT基因对 5 - FU的靶向性杀伤前列腺癌细胞增强作用和对前列腺癌自杀基因系统 (CD/ 5 -FC系统 )的放大效应奠定了基础。Objective To construct the recombinant expression plasmid pPSMA EP -UPRT including UPRT gene that is regulated by PSMA enhancer/ promoter . Methods By use of PCR, UPRT gene was amplified from E.coli JM109 genome. Then UPRT gene was cloned into the recombinant expression plasmid pPSMA enhancer/promoter -EGFP that is driven by prostate-specific membrane antigen promoter and enhancer. Results It was found that EGFP was digested by two restriction enzymes from the recombinant expression plasmid pPSMA enhancer/promoter -EGFP, and UPRT gene was linked with the recombinant expression plasmid by T 4 DNA ligase. We succeeded in constructing the recombinant expression plasmid pPSMA enhancer/promoter -UPRT. The recombinant plasmid sequences were verified, and the expression in vitro was measured by MTT. Conclusion The recombinant expression plasmid pPSMA enhancer/promoter -UPRT is regulated targetly by prostate-specific membrane antigen promoter and enhancer. It is of importance to us in studying the UPRT/5-FU for gene therapy of prostate cancer, especially suicide gene therapy (CD/5-FC system).

关 键 词：前列腺特异性膜抗原 启动子 增强子 尿嘧啶磷酸核糖转移酶基因 质粒 

分 类 号：R737.25[医药卫生—肿瘤]