人SAMHD1基因核心启动子区域的初步鉴定和分析

发布时间：2018-12-25 15:33

【摘要】：目的鉴定人不育-α-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Hep G2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。将扩增出的4个片段连入p MD19-T载体,双酶切鉴定后将DNA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DNA。PCR扩增后电泳结果显示已经扩增出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 GL3-399。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)。5'RACE结果显示SAMHD1在Hep G2细胞中转录起始位点位于-101。结论 SAMHD1的核心启动子位于翻译起始位点上游-101~-399区域,为深入研究肝细胞中SAMHD1转录调控机制奠定了基础。
[Abstract]:Objective to identify the core promoter region of human infertility 伪 -motif domain and histidine / aspartic acid residue double conjoined domain (SAMHD1) gene, and to study the transcriptional regulation mechanism of SAMHD1. Methods Genomic DNA,PCR was extracted from Hep G2 cells to amplify the 937667553399 bp fragment upstream of the translation initiation site of SAMHD1 gene. The four amplified fragments were inserted into the p MD19-T vector. The DNA bands were identified by double enzyme digestion and then recovered into the p GL3-Basic vector. The results were confirmed by double enzyme digestion and DNA sequencing. The luciferase expression vector containing four fragments was cotransfected with pRL-TK into Hep G2 cells. The promoter activity of luciferase reporter gene was detected and analyzed. The transcription initiation site of SAMHD1 in Hep G2 cells was identified by 5'RACE. Results the genomic DNA.PCR was extracted from Hep G cells and then amplified. The results showed that 937667553399 bp fragment had been amplified. The results of double enzyme digestion electrophoresis showed that the four amplified fragments were successfully inserted into p MD19-T vector. Construction of luciferase expression vectors p GL3-937,p GL3-667,p GL3-553 and p GL3-399. by double enzyme digestion and DNA sequencing The detection of luciferase reporter gene activity showed that the core promoter of SAMHD1 was located in the 0- 399 region (the previous base of ATG was -1). The 5'RACE results showed that the transcription initiation site of SAMHD1 in Hep G2 cells was -101. Conclusion the core promoter of SAMHD1 is located in the region of -101 ~ 399 upstream of the translation initiation site, which lays a foundation for further study on the mechanism of transcriptional regulation of SAMHD1 in hepatocytes.
【作者单位】：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;
【基金】：国家自然科学基金(编号:81372576) 安徽省自然科学基金(编号:1508085MH158) 安徽省博士后研究人员科研活动经费资助(编号:2015B066)
【分类号】：Q78

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