摘要
目的:构建survivin启动子调控的真核表达载体pSurp-EGFP,利用增强型绿色荧光蛋白(EGFP)检测该启动子在Hep-2细胞中的特异表达活性。方法:PCR扩增survivin启动子,置换pShuttle载体中的CMV启动子,构建质粒pSurp;分别双酶切pShuttle、pSurp和pEGFP-C1载体,构建分别携带survivin启动子和巨细胞病毒(CMV)启动子的真核表达载体pCMV-EGFP和pSurp-EGFP;脂质体法转染Hep-2细胞及血管内皮细胞ECV304,荧光显微镜下观察EGFP的表达。结果:成功扩增survivin基因启动子,构建了survivin基因启动子调控的真核表达载体pSurp-EGFP,用其转染两种细胞后,荧光显微镜下见Hep-2细胞发出较强的绿色荧光,而ECV304细胞没有表达。结论:成功克隆survivin启动子,其在Hep-2细胞具有较强的肿瘤特异性启动活性,为进一步开发肿瘤的特异性基因治疗奠定了实验基础。
Objective： To construct the eukaryotic expression vector pSurp-EGFP regulated by the survivin gene promoter and to detect the specific expression of the promoter in human laryngeal cancer Hep- 2 cells by green fluorescent protein assay. Methods ： Thesurvivin gene promoter was generated by polymerase chain reaction （PCR） and the CMV promoter of the pShuttle vector replaced by the survivin gene promoter to generate the plasmid pSurp. The three plasmids pShuttle, pSurp and pEGFP-C1 were respectively double-enzyme digested so as to produce the plasmids pCMV-EGFP and pSurp-EGFP carrying the CMV or survivin promoter. The purified pCMV-EGFP and pSurp-EGFP were transfected into Hep-2 cell and vascular endothelial cell ECV304 using liposome transfection reagent and the expressions of EGFP detected by the fluorescent microscope. Results： Thesurvivin gene promoter was successfully cloned by PCR, and thesurvivin gene promoter-regulated pSurp- EGFP was constructed. Green fluorescence wasobserved in Hep-2 cells but not in ECV304. Conclusion ： The high specific activity of the survivin gene promoter in Hep-2 cells that we successfully constructed attributes to the studies of tumor specific gene therapy.
出处
《医学研究生学报》
CAS
2008年第6期564-567,I0001,共5页
Journal of Medical Postgraduates
基金
国家自然科学基金资助项目(批准号:30371445)
