survivin基因启动子调控的HSV-tk真核表达载体的构建及在人喉癌细胞中的特异性表达

目的构建survivin基因启动子调控的人单纯疱疹病毒胸苷激酶(humansimplexvirus-thymidinekinase,HSV-tk)真核表达载体,检测其在人喉癌细胞中的表达。方法PCR克隆survivin基因启动子与tk基因,双酶切后分别插入pCI-neo真核表达载体中,酶切鉴定后用脂质体法转染喉癌细胞(Hep-2)和正常细胞(7702),RT-PCR法比较两种细胞tk基因的表达情况。结果成功构建了survivin启动子调控的tk基因真核表达载体pCI-neo/survivin-tk;并通过RT-PCR检测出survivin启动子调控的表达载体在喉癌细胞中有表达,而在正常细胞中无明显表达。结论survivin启动子具有肿瘤特异性,有可能解决喉癌基因治疗中的特异性杀伤问题。

Construction of an eukaryotic expression vector containing HSV-tk gene under the control of survivin promoter and specific expression in human laryngeal carcinoma cells.

Objective:To construct an eukaryotic expression vector containing HSV-tk gene under the control of survivin promoter and identify the expression of the vector in human laryngeal carcinoma cells. Methods:Survivin promotor and tk gene were amplified from plasmids by PCR,and were cloned into pCI-neo vector. After the eukaryotic expression vector was confirmed by restriction enzyme digestion, it was transfected into human laryngeal carcinoma cells(Hep-2) and human liver cells(7702) by liposome. RT-PCR was used to detect the tk gene in laryngeal carcinoma cells and normal cells. Results:The eukaryotic expression vector containing HSV-tk gene under the control of survivin promoter was successfully constructed. The result of RT-PCR indicated that tk gene was expressed in laryngeal carcinoma cells,but not in normal liver cells. Conclusion:Survivin promoter is tumor-specific and maybe useful in gene therapy of tumor.