發(fā)布時(shí)間：2018-12-25 15:33

【摘要】：目的鑒定人不育-α-基序結(jié)構(gòu)域和組氨酸/天冬氨酸殘基雙聯(lián)體結(jié)構(gòu)域包涵蛋白1(SAMHD1)基因的核心啟動(dòng)子區(qū)域,研究SAMHD1的轉(zhuǎn)錄調(diào)控機(jī)制。方法從Hep G2細(xì)胞中提取基因組DNA,PCR擴(kuò)增SAMHD1基因翻譯起始位點(diǎn)上游937、667、553、399 bp片段。將擴(kuò)增出的4個(gè)片段連入p MD19-T載體,雙酶切鑒定后將DNA條帶切膠回收,再連入p GL3-Basic載體中,雙酶切及DNA測(cè)序鑒定。將包含有4個(gè)片段的熒光素酶表達(dá)載體與pRL-TK共轉(zhuǎn)染Hep G2細(xì)胞,熒光素酶報(bào)告基因活性檢測(cè)并分析4個(gè)片段的啟動(dòng)子活性。5'RACE鑒定SAMHD1在Hep G2細(xì)胞中的轉(zhuǎn)錄起始位點(diǎn)。結(jié)果電泳結(jié)果顯示已經(jīng)從Hep G細(xì)胞中提取出基因組DNA。PCR擴(kuò)增后電泳結(jié)果顯示已經(jīng)擴(kuò)增出937、667、553、399 bp片段。雙酶切后電泳結(jié)果顯示成功將擴(kuò)增出的4個(gè)片段連入p MD19-T載體。雙酶切及DNA測(cè)序鑒定已成功構(gòu)建熒光素酶表達(dá)載體p GL3-937、p GL3-667、p GL3-553和p GL3-399。熒光素酶報(bào)告基因活性檢測(cè)顯示SAMHD1核心啟動(dòng)子區(qū)域位于0~-399區(qū)域(ATG前一個(gè)堿基為-1)。5'RACE結(jié)果顯示SAMHD1在Hep G2細(xì)胞中轉(zhuǎn)錄起始位點(diǎn)位于-101。結(jié)論 SAMHD1的核心啟動(dòng)子位于翻譯起始位點(diǎn)上游-101~-399區(qū)域,為深入研究肝細(xì)胞中SAMHD1轉(zhuǎn)錄調(diào)控機(jī)制奠定了基礎(chǔ)。
[Abstract]:Objective to identify the core promoter region of human infertility 偽 -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.
【作者單位】： 安徽醫(yī)科大學(xué)免疫學(xué)教研室;安徽醫(yī)科大學(xué)生物藥物研究所;
【基金】：國(guó)家自然科學(xué)基金(編號(hào):81372576) 安徽省自然科學(xué)基金(編號(hào):1508085MH158) 安徽省博士后研究人員科研活動(dòng)經(jīng)費(fèi)資助(編號(hào):2015B066)
【分類號(hào)】：Q78

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