人Cdc25B基因启动子报告基因载体的构建及转录活性分析

Construction of human Cdc25B gene promoter luciferase reporter gene vectors and analysis of transcription activity

目的构建人细胞分裂周期25家族蛋白B(Cdc25B)基因启动子(promoter)荧光素酶报告基因载体p GL3-Cdc25B-promoter,并检测其转录活性。方法应用欧洲启动子在线数据库(EPD)获得人Cdc25B基因5'端非编码区处包含转录起始位点在内的-2000^+100的DNA序列。以正常人全血基因组DNA为模板,利用PCR扩增该序列,并插入到p GL3-Basic荧光素酶报告基因载体上。通过设计不同引物,进一步构建系列缺失体,共获得7个不同长度启动子序列的报告基因载体。将其与内参质粒pRL-TK瞬时共转染He La细胞,通过双荧光素酶报告基因活性测定实验检测不同缺失体的转录活性。利用定点突变及RNA干扰(RNAi)探究预测的转录因子对Cdc25B转录活性的影响。结果构建的人Cdc25B基因启动子荧光素酶报告基因载体p GL3-Cdc25B-promoter经酶切鉴定及测序结果比对完全正确,将其转染He La细胞后,检测到荧光素酶高表达(P<0.05)。启动子系列缺失分析显示-160^-53片段包含基本核心启动子,与生物信息学的预测结果一致。定点突变及RNA干扰显示NF-YB转录因子对Cdc25B起正调控作用。结论成功构建了7个具有转录活性的人Cdc25B启动子缺失片段,确定了NF-YB是Cdc25B转录的一个重要调控因子,为进一步将其用于抗肿瘤药物的筛选与评价奠定了基础。

Objective To construct human Cdc25 B gene promoter luciferase reporter gene vectors containing different promoter sequences,and detect their transcription activity. Methods The 5＇UTR fragment（-2000 to ＋ 100） containing TSS of the human Cdc25 B gene was acquired from the Eukaryotic Promoter Database（EPD）. By designing different primers,the series of 5＇ deleted constructs were further constructed to obtain seven luciferase reporter gene vectors of different lengths. The recombinant plasmids were transiently co-transfected into He La cells with control vector pRL-TK before the transcriptional activity of Cdc25 B gene promoter was determined by the dual luciferase analysis. In addition,site-directed mutagenesis and RNA interference were applied to explore the effect of NF-YB on Cdc25 B promoter activity.Results The constructed p GL3-Cdc25 B-promoter vectors were proved by the restriction analysis and sequence alignment.After the recombinant plasmids were transfected,luciferase was highly expressed in He La cells. Promoter deletion analysis showed that the sequence between-160 to-53 contained the basal core promoter for human Cdc25 B gene,which was consistent with the prediction result obtained by bioinformatics. Site-directed mutagenesis and RNA interference indicated that NF-YB could interact with Cdc25 B gene promoter. Conclusion Seven human Cdc25 B gene promoter deletion fragments with transcriptional activity are successfully constructed. NF-YB is a crucial regulator of Cdc25 B transcription.