中文摘要：

目的：扩增survivin基因启动子,构建survivin启动子调控的真核表达载体,验证其在肿瘤细胞的特异性表达。方法：以Hep-2细胞基因组DNA为模板,PCR扩增survivin启动子;利用核酸内切酶双酶切sur-vivin启动子基因片段和pShuttle质粒,构建载体pSurp,酶切和测序鉴定;分别双酶切pSurp载体和pEGFP-C1载体,构建survivin启动子调控的真核表达载体pSurp-EGFP,脂质体转染Hep-2细胞及血管内皮细胞ECV304,荧光显微镜下观察EGFP的表达,Western blot方法验证该基因的蛋白表达。结果：成功扩增survivin基因启动子并构建survivin基因启动子调控的pSurp-EGFP真核表达载体。脂质体法转染Hep-2细胞和血管内皮细胞ECV304,在荧光显微镜下见Hep-2细胞发出较强的绿色荧光,而ECV304细胞没有绿色荧光;Western blot的结果也确证了EGFP蛋白仅在Hep-2细胞表达。结论：Survivin启动子的成功克隆与其真核表达载体的构建,为肿瘤的特异性基因治疗奠定了实验基础。

英文摘要：

Objective：By amplification of survivin gene promoter and construction of eukaryotic expression vector under the control of survivin promoter to prove its tumor specific expression.Methods：Using Hep-2 cell genomic DNA as template,the survivin gene promoter region was amplified by polymerase chain reaction.The amplified survivin gene fragment and pShuttle vector were double-enzyme digested to construct vector pSurp bearing survivin gene promoter,which was further confirmed by restriction analysis and DNA sequencing.Afterwards,plasmids pSurp and pEGFP-C1 were respectively double-enzyme digested to produce the vector pSurp-EGFP contolled by survivin promoter.The plasmid pSurp-EGFP was transfected into Hep-2 cell and vessel endothelial cell ECV304 using liposome reagent and expression of EGFP was detected by fluorescent microscope and Western blot.Results：Survivin gene promoter was cloned successfully by PCR method and eukaryotic expression vector pSurp-EGFP controlled by survivin gene promoter was constructed.Green fluorescence was observed in Hep-2 cells while not in ECV304 after transfected by pSurp-EGFP.Western blot analysis results showed that pSurp-EGFP expressed EGFP protein only in Hep-2 cells.Conclusion：Successfull survivin gene promoter cloning and construction of its expression vector might be the initial step of tumor specific gene therapy.