生存素启动子调控HSV1-TK真核表达载体的构建及其对A549肺癌细胞系的杀伤作用

Construction of a karyotic expression vector involving HSV-1 thymidine kinase under the control by survivin promoter and its potential use in ling gene therapy

目的:构建生存素(survivin,SURV)启动子调控的单纯疱疹病毒1型胸苷激酶(HSV1-TK)基因表达载体,检测该启动子的活性和特异性,观察该基因对大鼠体内肺癌生长的抑制功能。方法:酶切回收SURV启动子、CMV启动子;PCR扩增HSV1-TK基因;将上述片段与含有报告基因荧光素酶基因(Luciferase,Luc)的载体pGL3-Basic的多克隆位点连接,分别构建成为SURV、CMV启动子调控的HSV1-TK、Luc真核表达载体(pSURV-TK、pCMV-TK,pSURV-Luc、pCMV-Luc),用脂质体法将表达载体转染A549细胞系,蛋白质印迹法检测TK基因的表达,更昔洛韦细胞毒实验观察TK蛋白的酶活性,肿瘤生长抑制实验证实pSURV-TK的抑瘤功能。结果:成功地将SURV基因启动子、CMV启动子,HSV1-TK克隆到报告基因载体pGL3的多克隆位点,酶切鉴定和DNA序列分析无误,蛋白质印迹证实pSURV-TK在肺癌细胞A549中可特异性表达出TK蛋白,更昔洛韦细胞毒实验表明表达的TK蛋白具有酶活性,并可有效抑制体内肺癌的生长。结论:SURV启动子调控的TK基因治疗载体,可特异性调控TK基因在A594肺癌细胞的表达并抑制肺癌细胞的生长,为应用自杀基因治疗肺癌提供了新的途径。

Objective： To construct a lung cancer specific the herpes simplex virus 1 thymidine kinase（HSV1-TK） expression vector regulated by survivin promoter（SURV） and detect its activation and potential to inhibit the grow of lung cancer in vivo.Methods： Plasmid pGL3-TK was obtained by substituting the luciferase cassette with a PCR product corresponding to TK with flanking Nco Ⅰ and Xho Ⅰ sites.To obtain the pSURV-TK and pCMV-TK two versions,the SURV and CMV cassettes were excised with Bgl Ⅱ and Hand Ⅲ and cloned into the respective sites of the vector.Recombinant plasmid pSURV-TK and pCMV-TK were transfected into survivin positive or negative cell lines by the means of lipofectamine.The expression of HSV1-TK was tested by Western Blotting,Ganciclovir cytotoxicity assay to evaluate the activation of SURV and in vivo tumorigenesis experiments to explore the potential using this vector to treat lung cancer.Results： The vector pSURV-TK has been successfully constructed which can not only express the TK protein indentified by Western Blotting,but also show its enzyme activation related to the concentration of ganciclovir and suppression of tumor growth in vivo.Conclusion： Survivin promoter can effectively drive the HSV1-TK to express.The expression of TK gene will be confined just within the site of tumor.This presented a new way of employment sucide genes for treatment of lung cancer.