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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 determine the active region of cnr1 in ischemic tolerance of spinal cord according to the transcriptional activity of different parts of the promoter sequence of cannabinoid receptor 1 (cnr1) using dual luciferase reporter gene system The study of transcriptional regulation lay the foundation. METHODS: The nucleotide sequence of about 1800 bp upstream of the transcription initiation site of cnr1 gene was obtained from NCBI. Six truncated PCR primers of different lengths were designed according to the distance of 300 bp. The DNA of genomic DNA of human whole blood was The truncated fragment of cnr1 promoter region was amplified and cloned into luciferase reporter plasmid pGL3-Basic. The recombinant plasmids containing different truncated bodies were respectively transfected into Hela, Jurket and A549 cells for luciferase activity assay. According to the different truncated transcription activity test results, determine cnr1 promoter activity region. Results: Six truncates of different lengths (1800, 1500, 1200, 900, 600 and 300 bp) in the cnr1 promoter region were successfully cloned into the luciferase reporter gene pGL3-Basic plasmid. The detection of luciferase activity in Hela, Jurket and A549 in all three cell lines showed that the 600 bp truncated truncated transcription activity was the strongest. CONCLUSION: The reporter gene of cnr1 promoter was successfully constructed and the precursor region of -600 bp to -200 bp was found to be the active region of cnr1 promoter, which laid the foundation for further study on the transcriptional regulation of cnr1.