Survivin基因启动子调控的肿瘤特异性表达载体的构建

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。

Construction of a tumor specific expression vector under the control of survivin gene promoter

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.