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

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UPRT基因真核表达重组质粒(pPSMAenhaocer／promoter-UPRT)。方法 采用PCR技术从大肠杆菌JMpPSMAenhaocer／promoter-UPRT109基因组中扩增UPRT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pEGFP-1的质粒。利用重组质粒pPSMAenhaocer／promoter-UPRT)转染LNcap细胞，MTT法检测5-氯尿嘧啶(5-FU)对转染LNcap细胞存活率的影响。结果 质粒pPSMAenhaocer／promoter-EGFP双酶切去除EGFP，连接上UPRT基因，成功构建pPSMAenhaocer／promoter-UPRT,并转染LNcap细胞。使LNcap细胞对5-FU的杀伤敏感性大大提高。结论 pPSMAenhaocer／promoter-UPRT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／5一FC系统)的放大效应奠定了基础。

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

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).