CD226基因表达调控的研究

发布时间：2018-04-15 23:38

本文选题：CD226 + 启动子　；参考：《第四军医大学》2005年博士论文

【摘要】：目的:研究人CD226基因表达调控的规律,确定CD226基因启动子的位置,研究细胞因子、刺激剂和转录因子对其的调节作用。方法:①从50ng/ml PMA刺激48小时的Jurkat细胞中提取mRNA,采用5′-RACE的方法经过反转录和巢式PCR确定人CD226基因的转录起始点。②从GenBank中获取CD226基因的上游调控序列,利用www.ifti.org中的转录因子扫描程序,分析可能存在的转录因子结合位点,用手工比对的方法剔除分析结果中可能存在的假阳性,筛选可能性比较大的转录因子进行功能研究。③从正常人外周血中提取基因组DNA,以GenBank中获得的序列为模板设计引物,克隆了长约2kb的CD226基因上游调控序列。在此基础上,制备了不同的截断体,连入pGL3荧光素酶报告基因表达载体,转染Jurkat细胞48小时后检测荧光素酶活性,确定CD226的启动子的位置。④将含有CD226基因启动子序列的报告基因载体转染Jurkat细胞,用50ng/ml PMA和0.5μg/ml A23187分别刺激4小时和1小时,在转染48小时后检测荧光素酶活性,观察PMA和A23187对CD226基因启动子的调控作用。⑤把两个启动子间可能存在的负调控元件(NRE)通过PCR的方法,克隆到CMV启动子的下游,DNA测序正确后将CMV-NRE
[Abstract]:Aim: to study the regulation of human CD226 gene expression, determine the position of CD226 gene promoter, and study the regulatory effects of cytokines, stimulators and transcription factors.Methods MRNAs were extracted from Jurkat cells stimulated by 50ng/ml PMA for 48 hours. The upstream regulatory sequence of CD226 gene was obtained from GenBank by reverse transcription and nested PCR by 5'-RACE.Using the transcription factor scanning program in www.ifti.org, the possible transcription factor binding sites were analyzed, and the false positives in the analysis results were eliminated by the method of manual comparison.Functional study on the screening of transcription factors. 3 the genomic DNAs were extracted from the peripheral blood of normal subjects. Using the sequences obtained from GenBank as template primers, the upstream regulatory sequence of CD226 gene was cloned.On this basis, different truncation bodies were prepared and inserted into pGL3 luciferase reporter gene expression vector. After 48 hours of transfection into Jurkat cells, luciferase activity was detected.The reporter gene vector containing the promoter sequence of CD226 gene was transfected into Jurkat cells. The promoter was stimulated with 50ng/ml PMA and 0.5 渭 g/ml A23187 for 4 hours and 1 hour, respectively. The luciferase activity was detected after 48 hours of transfection.Observation of the regulatory effect of PMA and A23187 on the promoter of CD226 gene ..5 the possible negative regulatory element between the two promoters was cloned into the downstream of the CMV promoter by PCR, and the CMV-NRE was sequenced correctly.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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