中文摘要：

目的构建表达血清类黏蛋白1样蛋白3（ORMDL3）基因第一内含子中新启动子区的质粒,并研究其功能特征。方法以人基因组DNA为模板,PCR扩增ORMDL3基因第一内含子内新转录起始位点上下游776bp启动子区片段,酶切并连接此片段至pGL3-basic载体,构建含有ORMDL3基因第一内含子中新启动子区的基因重组体,并转染人胚肾293细胞。荧光素酶检测报告基因启动子的活性,计算相对活性单位。生物信息学方法分析转录因子结合位点。结果成功构建含有新启动子的重组质粒。与pGL3-basic空载体相比,新启动子活性明显增强,并定位于转录起始位点-363bp至-63bp之间,且该区域存在Sp1、GATA-1等转录因子的结合位点。结论 ORMDL3基因第一内含子中新转录起始位点上游具有启动子活性,在-363bp至-63bp区域存在转录调控元件,且Sp1、GATA-1等转录因子可能参与其转录调控。

英文摘要：

Objective To construct a luciferase reporter plasmid containing new promoter in the first intron of orosomucoid 1-like 3（ORMDL3） gene and evaluate its functional characteristics.Methods The 776 bp fragment in the new transcription start site of the first intron of ORMDL3 gene was amplified by PCR with human genomic DNA as a template and was directionally cloned into pGL3-basic plasmid to construct a gene recombinant containing the new promoter in the first intron of ORMDL3 gene.Transfection of HEK-293 cells with the promoter-driven luciferase construct was performed to induce luciferase gene expression and calculate the relative luciferase activity unit.The binding site of transcription factor was analyzed by bioinformatics software.Results It was successful to construct the recombinant plasmid containing the promoter in the first intron of ORMDL3 gene.Compared with pGL3-basic plasmid,the new promoter activity was increased and located in front of new transcription start sites between-363 bp and-63 bp,where included binding sites of Sp1 and GATA-1 transcription factors.Conclusion The upstream region of new transcription start site in the first intron of ORMDL3 gene has promoter activity.The region between-363 bp and-63 bp exists the regulatory elements area,and the Sp1 and GATA-1 transcription factors may be involved in this transcriptional regulation.