| SP1调控PIWIL1基因启动子转录活性研究 |
| 投稿时间：2014-07-13 修订日期：2015-03-23 点此下载全文 |
| 引用本文：叶丽萍,顾彬彬,方从诚,朱敏,毛鑫礼,王超.SP1调控PIWIL1基因启动子转录活性研究[J].医学研究杂志,2016,45(6):133-137 |
| DOI：
10.11969/j.issn.1673-548X.2016.06.034 |
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| 中文摘要:目的 克隆转录因子SP1编码序列，构建真核表达质粒PCDNA3.1-SP1，并研究其对PIWIL1基因转录活性的影响。方法 RT-PCR法从正常人外周血总RNA中钓取SP1基因的编码序列，插入至真核表达载体PCNDA3.1（+），酶切及测序鉴定重组质粒。Western blot法分别验证重组表达载体的在COS-7细胞中的表达效应。分别将活性最高的PIWIL1基因启动子报告基因截短体（即核心启动子区域）、活性最低的PIWIL1基因启动子报告基因截短体，与SP1重组质粒共转染COS-7细胞，利用Dual-Luciferase reporter assay system测定不同启动子活性区的PIWIL1基因转录活性的改变，同时应用光辉霉素A下调SP1的表达来进一步验证SP1对PIWIL1活性的影响。结果 酶切及测序鉴定结果显示SP1表达质粒构建成功，Western blot法结果表明重组SP1表达载体能有效表达于COS-7细胞，过表达SP1可明显提高PIWIL1不同活性区的启动子的活性（P<0.05）。结论 外源性SP1真核表达载体转染能有效表达于COS-7细胞，并对PIWIL1的转录活性起着上调的作用，提示SP1可能是PIWIL1转录活性的正调控元件之一。 |
| 中文关键词:转录因子SP1 载体构建 启动子 转录调控 |
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| Transcription Factor Sp1 Regulates the Promoter Activity of PIWIL1 Gene |
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| Abstract:Objective To construct the eukaryotic expression vector of PCDNA3.1-sp1 and investigate the effects of SP1 on PIWIL1 transcriptional activity. Methods Using the the total RNA of lymphocyte of healthy human peripheral blood as template, the human SP1 CDNA was amplified by RT-PCR and inserted into the eukaryotic expression vector PCDNA3.1+. The recombinant plasmid was identified by sequencing and restriction endonuclease digestion. Western blot confirmed the the expression plasmid PCDNA3.1-SP1 can increase protein level. PCDNA3.1-SP1 was co-transfected into COS-7 cells with the highest activity of PIWIL1 gene promoter and the lowest activity of PIWIL1 gene promoter reporter respectively. The transactivity of PIWIL1 promoter was assayed by luminometer. These results were proven by blocking SP1 using Mithramycin A. Results The recombinant plasmid was confirmed by sequencing and restriction,endonuclease digestion,Western blot. Over expression of SP1 can improve the activity of PIWIL1 promoter. Conclusion The eukaryotic expression plasmid of PCDNA3.1-SP1 has been successfully constructed, and SP1 can increase PIWIL1 transcription activity. |
| keywords:Transcription factor SP1 Construct expression vector Promoter Transcriptional regulation |
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