人DRD1基因启动子区克隆和双荧光素酶报告基因系统的构建

Constructing the dual luciferase reporter vector containing human DRD1 promoter region

背景:多巴胺受体启动子区多态性位点的改变会影响受体的表达量,进而影响多巴胺能神经递质的作用,导致相关疾病的发生。目的:通过克隆人DRD1基因的启动子区,构建含有该基因启动子序列的双荧光素酶报告基因载体并检测其活性,为研究DRD1基因的转录调控提供基础工具。方法:以人静脉血基因组DNA为模板,通过PCR扩增DRD1基因启动子序列并克隆至pG M-T载体,测序验证无误后酶切连接至萤火虫荧光素酶报告基因质粒pG L3-Basic,构建含有DRD1基因启动子的荧光素酶报告基因载体,采用阳离子脂质体法与内对照质粒pG L3-TK共转染HEK293细胞同时以不含启动子的pG L3-Basic载体与内对照质粒pG L3-TK共转染作为阴性对照组,化学发光仪检测相对荧光强度。结果与结论:(1)通过酶切和测序验证重组荧光素酶报告基因载体的DRD1基因启动子区序列正确;(2)与阴性对照组相比,该重组载体转染HEK293细胞后具有转录活性;(3)成功构建了含有DRD1基因启动子区的荧光素酶报告基因载体,为研究该基因的转录调控提供了基础工具。

BACKGROUND： The polymorphisms of dopamine receptor in promoter region will affect the expression of the receptor, thereby affecting the dopaminergic neurotransmitter, finally lead to related diseases. OBJECTIVE： To construct the dual luciferase reporter vector containing human DRD1 promoter region and determine its activity, which could provide the basic tool for studying the transcriptional regulation of DRD1 gene. METHODS： DRD1 promoter sequence was amplified by PCR using the human blood genomic DNA and cloned into pG M-T vector. After sequencing, the correctly constructed vectors were ligated to the firefly luciferase reporter plasmid pG L3-Basic. The cloned p GL3-Basic vectors were transfected into HEK293 using cationic liposome method. In the meanwhile, PGL3-Basic vector with no promoter was co-transfected with p GL3-TK plasmid as negative control group. The relative fluorescence intensity was measured by chemiluminescence. RESULTS AND CONCLUSION：（1） Recombinant luciferase reporter gene vectors were confirmed by restriction analysis and sequencing.（2） Compared with the negative control group, the HEK293 cells transfected by recombinant vectors presented transcriptional activity.（3） In conclusion, luciferase reporter gene vectors containing DRD1 promoter region are successfully constructed and can provide the basic tool for further study on the transcriptional regulation of DRD1.