人SAMHD1基因核心启动子区域的初步鉴定和分析.docxVIP

人SAMHD1基因核心启动子区域的初步 鉴定和分析Lir. 鉴定和分析 Lir. 张栋 李苗苗 董阳刘星赞 应松成 方圣云 沈玉先 安徽医科大学免疫学教研室安徽医科大学生物药物 研究所 摘要： 目的鉴定人不育-?-基序结构域和组氨酸/天冬氨酸残基双联体结构域包涵蛋 白1 (SAMHD1)基因的核心启动子区域，研究SAMHD1的转录调控机制。方法从 HepG2细胞中提取基因组DNA, PCR扩增SAMHD1基因翻译起始位点上游937、667、 553、399 bp片段。将扩増出的4个片段连入p MD19-T载体，双酶切鉴定后将 DMA条带切胶回收，再连入p GL3-Basic载体中，双酶切及DNA测序鉴定。将包 含有4个片段的荧光素酶表达载体与pRL-TK共转染Hep G2细胞，荧光素酶报告 基因活性检测并分析4个片段的启动子活性。5 RACE鉴定SAMHD1在Hep G2细 胞中的转录起始位点。结果 电泳结果显示已经从Hep G细胞中提取出基因组 DNAo PCR扩増后电泳结果显示已经扩増出937、667、553、399 bp片段。双酶 切后电泳结果显示成功将扩增出的4个片段连入p MD19-T载体。双酶切及DNA 测序鉴定已成功构建荧光素酶表达载体P GL3-937、p GL3-667、p GL3-553和p GL3-399o荧光素酶报告基因活性检测显示SAMHD1核心启动子区域位于0 399区 域(ATG前一个碱基为-1) o 5 RACE结果显示SAMHD1在Hep G2细胞中转录起始 位点位于T01。结论SAM1ID1的核心启动子位于翻译起始位点上游-10T399区域, 为深入研究肝细胞中SAMHD1转录调控机制奠定了基础。 关键词： SAMHD1; 核心启动了; 转录起始位点; 转录调控; 作者简介：张栋，男，硕士研究生； 作者简介：应松成，男，副教授，硕士生导师，责任作 者;E-mail:yingsc@ahmu. ; 作者简介：方圣云，男，教授，博士生导师，责任作者， E-mail: sfang@unmryland. edu 收稿日期：2017-05-19 基金：国家自然科学基金(编号 Identification and analysis of the core promoter region of human SAMHD1 gene Zhang Dong Li Miaomiao Dong Yang Dept of Immunology, Anhui Medical University; Abstract： Objective To identify the core promoter region of human antiviral SAMHD1 gene. Methods Genomic DNA was extracted from Hep G2 cells, then 937, 667, 553 and 399 bp fragments before the translation start site of SAMIID1 gene were amplified by PCR from genomic DNA. Four fragments were inserted into the p MD19-T vector. After enzyme digestion and electrophoresis, DNA bands were separated and ligated into p GL3-Basic vector. The p GL3 plasmids containing four fragments were further identified by double enzyme digesti on and DNA sequcnci ng. The luciferase expression vcc tor con twining four fragments was co-transfected with pRL-TK into Hep G2 cells and the promoter activities of four fragments were analyzed by luciferase assay. The trainscription initiation site of SAMHD 1 in Hep G2 cells was identified by 5’ RACE. ResuIts Electrophoretic resuIts showed that genomic DNA had been successfully cxtracted