基于双荧光素酶报告基因系统的人源SREBP1基因启动子的活性研究

Study on the activity of human SREBP1 promoter based on dual luciferase reporter system

目的构建固醇调节元件结合蛋白1(SREBP1)启动子双荧光素酶报告基因表达载体,为研究针对SREBP1靶点的天然活性抑制剂筛选奠定基础。方法利用生物信息学软件,对SREBP1基因5′端上游5 000 bp序列进行启动子预测与分析。应用Gibson Assembly方法构建启动子双荧光素酶载体,并将构建成功的pGL3-SREBP1-pro重组质粒和内参质粒p RL-SV40共转染肝癌HepG2细胞并给予天然抑制剂大黄素,检测SREBP1转录活性的抑制效果。结果 SREBP1基因5′端上游2 000 bp区域内有启动子特征序列,存在转录因子结合位点;重组质粒经酶切及测序鉴定证实为阳性克隆,瞬时转染肝癌HepG2细胞后经双荧光素酶报告基因系统检测,所克隆的片段序列具有启动子活性,该活性可被大黄素所抑制。结论成功构建了pGL3-SREBP1-pro双荧光素酶报告基因表达载体,并证实其活性,可为进一步用于针对SREBP1靶点的天然活性抑制剂筛选奠定基础。

Objective To construct a dual luciferase reporter expression vector of sterol regulatory element-binding protein 1 (SREBP1) gene and to screen natural active inhibitors for SREBP1 targets. Methods The bioinformatics approaches were applied to predict and analyze the promoter region of SREBP1 from 5′ upstream 5 000 bp regulatory region. The dual luciferase reporter expression vector was constructed by Gibson Assembly method. The recombinant plasmid pGL3- SREBP1-pro and control plasmid pRL-SV40 were co-transfected into HepG2 cells. The inhibitory effects of the transcriptional activity of SREBP1 with or without natural active inhibitors were detected. Results There were promoter sequences and transcription factor binding sites in the 5′ upstream 2 000 bp region of SREBP1. The recombinant plasmid was confirmed as a positive clone by enzyme digestion and sequencing. The plasmid was transiently transfected in HepG2 cells to detect promoter activity of SREBP1 by dual luciferase reporter system, and transcriptional activity of SREBP1 was further verified by natural active inhibitors. Conclusion The pGL3-SREBP1-pro dual luciferase reporter expression vector is successfully constructed and its activity is confirmed, which can be further used for the screening of natural active inhibitors for SREBP1 targets.