【摘要】 目的　探讨由Myc Max二聚体作用序列替代单纯疱疹病毒 (HSV)胸腺嘧啶 (TK)核苷激酶启动子第 3区后 ,形成的嵌合结构在c myc过表达细胞中的特异启动活性。方法　通过聚合酶链反应(PCR)产物克隆和一系列亚克隆技术 ,构建由Myc Max作用序列与HSV TK启动子部分区域组成的嵌合启动子 ,将荧光素酶报告基因置于其控制之下 ,构建成荧光素酶表达质粒 ,瞬时转染已经Northernblot证实的c myc过表达和低表达细胞系 ,检测细胞提取物中的荧光素酶活性。结果　在c myc过表达的PC 2和PC 7细胞中 ,嵌合启动子 (Mpr)启动表达的荧光素酶活性分别为 ( 81966±4 3 2 3 8)和( 70 5 63±2 2 4 3 5 )相对发光值 (RLU) ,为剩余TK部分启动子 (Epr)启动活性的 78倍和 15 0倍 ,并是完整TK启动子 (TKpr)的 6 7倍和 1 7倍 ;在c myc低表达细胞中 ,Mpr启动活性很弱 ,其启动的荧光素酶活性为 ( 4 3 1± 73 )RLU ,与Epr启动的荧光素酶活性 ( 60 1±14 1)RLU近似。结论　由Myc Max蛋白作用序列与TK部分启动子组成的嵌合结构在c myc过表达细胞中具有特异启动活性。更多还原

【Abstract】 Objective To investigate the specific activity of a hybrid promoter which is constructed by replacing the third domain of herpes simplex virus (HSV) thymidine kinase (TK) promoter with the Myc Max response elements Methods Myc Max response elements were ligated with a third domain deleted HSV TK promoter by cloning and subcloning PCR products Then a luciferase expressing plasmid, in which the luciferase gene was put under the control of hybrid promoter, was constructed and transfected transiently into the cell lines which had been demonstrated to be c myc over or low expressing by Northern blot hybridization The luciferase activities in these cells were detected Results In c myc over expressing cells, the hybrid promoter (Mpr) led to high levels of (81 966±43 238) relative light units (RLUs) in PC 2 cells and (70 563±22 435) RLUs in PC 7 cells, which were 78 and 150 fold higher than those coming from the third domain deleted TK promoter (Epr), and also 6 9 and 1 7 fold higher than the activities controlled by TK promoter However, Mpr showed a very low activity in c myc low expressing cells, in which the luciferase activity was (431±73) RLUs, similar to (601±141) RLUs produced by Epr Conclusion The activity of hybrid promoter, which is composed of Myc Max response elements and the third domain deleted TK promoter, possesses cell type specificity for c myc overexpressing cells更多还原