目的: 构建带有survivin启动子的pGL3Basic真核表达载体，探讨survivin启动子在HeLa细胞中的特异表达活性。 方法: 采用PCR技术扩增survivin启动子，插入pGL3Basic载体，构建携带survivin启动子的pGL3Basic真核表达载体（pGL3Basic/ Surp）。纯化pGL3Basic/ Surp质粒，用脂质体法转染HeLa细胞和正常血管内皮细胞ECV304，48 h后收集转染细胞与荧光素酶底物反应，检测荧光素酶活性。 结果: 成功克隆1 kb survivin基因启动子，并构建了携带有survivin基因启动子的pGL3Basic真核表达载体，转染后的Hela细胞荧光素酶活性为2074.2±78.5，而ECV304荧光素酶活性为9.7±1.1。 结论： 成功克隆的survivin启动子在HeLa细胞中表现出较高的肿瘤特异性活性，为进一步开发肿瘤的靶向基因治疗奠定了基础

Objective : To construct pGL3Basic eukaryotic expression vector containing survivin promoter gene and explore the activity of this survivin promoter in Hela cells. Methods: The survivin gene promoter was amplified by polymerase chain reaction and cloned into pGL3Basic vector to construct pGL3Basic eukaryotic expression vector containing survivin gene promoter (pGL3Basic/Surp). The purified pGL3Basic/Surp was transiently transfected into HeLa cell and vessel endothelial cell line EVC304 using liposome transfection reagent and the activity of survivin gene promoter was determined by adding luciferase substrate into transfected cells 48 h later. Results: About 1 kb gene fragment was amplified by PCR method from Hela cell genomic DNA and pGL3Basic/Surp vector was constructed successfully. The activity of luciferase reporter gene was 2074.2±78.5 in Hela and 9.7±1.1 in EVC304 48 h after transfection of pGL3Basic/surp vector. Conclusion: The high specific activity of constructed survivin promoter eukaryotic expression vector might be a potential therapeutic reagent for the treatment of malignant tumor.

国家自然科学基金资助( 30371445); 陕西省社发计项目（2003K10G44）