人大麻素受体启动子真核报告质粒的构建及活性分析

Construction and analysis of the luciferase reporter vectors of human cnr1 promoter

目的:利用双荧光素酶报告基因体系,根据大麻素受体1(cannabinoid receptor 1,cnr1)启动子序列不同部位的转录活性来初步确定其活性区域,为脊髓缺血耐受保护中cnr1高表达的转录调控的研究奠定基础。方法:从NCBI中获取cnr1基因转录起始点5’端向上约1800 bp的核苷酸序列,按照300 bp的距离间隔,设计6个不同长度的截短体PCR引物,以人全血基因组DNA为模板,分别扩增cnr1启动子区域的截短体片段,并克隆入荧光素酶报告基因pGL3-Basic质粒中。将含有不同截短体的重组质粒分别转染Hela、Jurket和A549细胞后行荧光素酶活性检测。根据不同截短体转录活性检测结果,确定cnr1启动子活性区域。结果:成功将cnr1启动子区6个不同长度(1800、1500、1200、900、600和300 bp)的截短体克隆入荧光素酶报告基因pGL3-Basic质粒。在3种细胞系Hela、Jurket和A549的荧光素酶活性检测均显示600 bp的截短体转录活性最强。结论:成功构建了cnr1启动子的报告基因重组质粒,初步证实-600 bp到-200 bp区为cnr1的启动子的活性区域,从而为进一步研究cnr1的转录调控奠定了基础。

Objective：To identify the active region of cnr1 promoter gene in dual-luciferase reporter assay.Methods：According to the cnr1 sequences from NCBI,the six truncated bodies with different length were cloned into luciferase reporter gene pGL3-Basic respectively.Then the recombinant constructs were transfected into PGL3-Basic plasinid.On the basis of transcriptional activity of every truncated body in dual-luciferase reporter assay,the activation region of cnr1 promoter was defined.Results：The six recombinant plasmids containing truncated bodies（1800 bp,1500 bp,1200 bp,900 bp,600 bp and 300 bp） of cnr1 were obtained successfully.Among the six truncated bodies of cnr1 promoter in Hela,Jurket and A549 cell lines,the transcriptional activity of pGL3-600（600 bp） was strongest.Conclusion：The active region of cnr1 promoter is from-200 bp to-600 bp of the gene cnr1.The results laid the experimental foundation to further study the transcriptional regulation of cnr1.