人SAMHD1基因核心启动子区域的初步鉴定和分析

Identification and analysis of the core promoter region of human SAMHD1 gene

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。

Objective To identify the core promoter region of human antiviral SAMHD1 gene. Methods GenomicDNA was extracted from HepG2 cells, then 937, 667, 553 and 399 bp fragments before the translation start site of SAMHD1 gene were amplified by PCR from genomic DNA. Four fragments were inserted into the pMD19-T vector. After enzyme digestion and electrophoresis, DNA bands were separated and ligated into pGI3-Basic vector. The pGL3 plasmids containing four fragments were further identified by double enzyme digestion and DNA sequencing. The luciferase expression vector containing four fragments was co-transfected with pRL-TK into HepG2 cells and the promoter activities of four fragments were analyzed by luciferase assay. The transcription initiation site of SAMHD1 in HepG2 cells was identified by 5＇RACE. Results Electrophoretic results showed that genomic DNA had been successfully extracted from HepG cells. The results of electrophoresis after PCR amplification showed that 937, 667, 553 and 399 bp fragments had been amplified. The results of electrophoresis showed that four fragments were successfully inserted into pMD19-T vector. Double enzyme digestion and DNA sequencing confirmed that luciferase expression vectors pGI_3-937, pGI_3-667, pGI_3-553 and pGI3-399 were successfully constructed. Luciferase ac- tivity analysis showed that the SAMHD1 core promoter region was located in the 0 - -399 region（ the first base be- fore ATG was set as -1 ）. 5＇RACE results showed that the transcription initiation site of SAMHD1 in HepG2 cells was located at - 101. Conclusion The core promoter of SAMHD1 is located at - 101 - - 399 region, which lays a foundation for further study of SAMHD1 transcriptional regulation in hepatocytes.