摘要
目的:扩增survivin基因启动子并构建带有survivin启动子的pEGFPN1真核表达载体,检测survivin基因启动子在HeLa细胞中的特异表达活性.方法:①以HeLa细胞基因组DNA为模板,PCR扩增survivin启动子,回收扩增产物并插入pGEMTEasy载体(T/Surp),测序鉴定.②分别双酶切TSurp载体和不含CMV启动子的pEGFPN1载体,回收surviv启动子酶切片段及pEGFPN1酶切线性化片段,连接回收产物构建携带survivin启动子pEGFPN1真核表达载体(pEGFN1/Surp),酶切鉴定.③转染HeLa细胞及正常血管内皮细胞ECV304,在荧光显微镜下观察GFP的表达.结果:酶切及测序证实成功扩增survivin基因启动子,并构建了携带有suvivin基因启动子的pEGFPN1真核表达载体.转染HeLa细胞和正常血管内皮细胞ECV304,在荧光显微镜下见HeLa细胞发出较强的绿色荧光,而在ECV304细胞未见绿色荧光结论:成功克隆的肿瘤特异性survivin启动子在HeLa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.
AIM: To molecular clone survivin gene promoter and detect the activity of survivin promoter in Hela cells. METHODS: (1) Approximately 1 kb of the survivin gene promoter region was generated by polymerase chain reaction using HeLa cell genomic DNA as a template and cloned into the vector pGEM-T-easy to generate the plasmid survivin-T-easy (T/Surp) .The plasmid was transformed into JM109 E.coli competent cells and positive clone was selected .The survivin gene promoter was further confirmed by DNA sequence analysis. (2)The survivin gene promoter in T vector was cut with SacⅠ and HindⅢ enzyme and received ,then religated with pEGFP-N1 vector which not include CMV promoter to generate the plasmid pEGFP-N1/ Surp. (3) The purified pEGFP-N1/ Surp was transfected into HeLa cell and ECV304,then detected the expression of GFP by fluorescent microscope. RESULTS: A 1 kb of survivin gene promoter was cloned successfully by PCR method from HeLa cell genomic DNA and pEGFP-N1 bearing survivin gene promoter was constructed. Green fluorescence was observed in HeLa cell while not in ECV304. CONCLUSION: The high specific activity of survivin gene promoter that we constructed is expected to be useful in tumor specific gene therapy.
出处
《第四军医大学学报》
北大核心
2005年第12期1063-1065,共3页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30371445)
