人二硫键氧化还原酶类似蛋白基因启动子克隆和上游抑制元件分析
投稿时间:2016-03-28  修订日期:2016-04-09  点此下载全文
引用本文:陈淑芹,徐宜兰,白宁宁,张菁,方启晨,贾伟平.人二硫键氧化还原酶类似蛋白基因启动子克隆和上游抑制元件分析[J].医学研究杂志,2016,45(9):76-81
DOI: 10.11969/j.issn.1673-548X.2016.09.020
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作者单位E-mail
陈淑芹 200233 上海交通大学附属第六人民医院内分泌代谢科、上海市糖尿病重点实验室、上海市糖尿病研究所、上海市糖尿病临床医学中心  
徐宜兰 200233 上海交通大学附属第六人民医院内分泌代谢科、上海市糖尿病重点实验室、上海市糖尿病研究所、上海市糖尿病临床医学中心  
白宁宁 200233 上海交通大学附属第六人民医院内分泌代谢科、上海市糖尿病重点实验室、上海市糖尿病研究所、上海市糖尿病临床医学中心  
张菁 200233 上海交通大学附属第六人民医院内分泌代谢科、上海市糖尿病重点实验室、上海市糖尿病研究所、上海市糖尿病临床医学中心  
方启晨 200233 上海交通大学附属第六人民医院内分泌代谢科、上海市糖尿病重点实验室、上海市糖尿病研究所、上海市糖尿病临床医学中心 qcfang@sjtu.edu.cn 
贾伟平 200233 上海交通大学附属第六人民医院内分泌代谢科、上海市糖尿病重点实验室、上海市糖尿病研究所、上海市糖尿病临床医学中心  
基金项目:上海市自然科学基金资助项目(15ZR1431700)
中文摘要:目的 克隆DsbA-L基因启动子,并对其活性进行初步分析。方法 以人基因组DNA为模板,利用PCR技术扩增DsbA-L基因5’端2115bp片段(从翻译起始点ATG上游-2128~-14区域)。将PCR产物插入荧光素酶报告基因载体pGL3-basic。随后,构建5’端系列缺失的报告基因质粒。将构建的一系列质粒转染3T3-L1和HEK 293细胞,检测荧光素酶活性。利用在线软件MAPPER预测转录调控关键序列潜在的转录因子结合位点。通过Real-time PCR比较这些转录因子在肥胖和正常人脂肪组织中的表达。结果 成功构建人DsbA-L启动子报告基因质粒。系列缺失分析表明-2128~-1302区域是影响转录活性的关键序列。MAPPER软件预测此区域可能与一些转录因子结合。比较这些转录因子在肥胖患者和正常人脂肪组织中的表达情况,发现其中NF-κB和FOXO1在肥胖患者中表达显著增加,进一步推测NF-κB和FOXO1可能是人DsbA-L基因启动子上游的调控抑制因子。结论 成功构建人DsbA-L基因启动子系列缺失质粒,初步分析了其上游可能存在的关键调控元件,为进一步的转录调控研究奠定了基础。
中文关键词:二硫键氧化还原酶类似蛋白  转录调控  启动子
 
Cloning of the Human Disulfide-bond-A oxidoreductase-like Protein Gene Promoter and Identification of the Upstream Repressor
Abstract:Objective To clone the promoter of human Disulfide-bond-A oxidoreductase-like protein, and to analysis its activity preliminary. Methods A 2115bp fragment containing the 5'-flanking region of human Disulfide-bond-A oxidoreductase-like Protein gene(-2128 to -14 related to ATG) was amplified by PCR using human genomic DNA as the template. The PCR product was inserted in to reporter vector pGL3-basic. Then a series of 5' deletion reporter constructs was generated. The various firefly luciferase reporter plasmids were transfected into 3T3-L1 and HEK293 cells, then luciferase activity was assessed. Online software MAPPER was used to predict the potential transcriptional factor of this region. Comparison of the expression levels of these transcription factor in the adipose tissue of the obese and non-obese people was conducted to deduce the key regulatory elements. Results The promoter of human Disulfide-bond-A oxidoreductase-like protein was successfully cloned. Deletion analysis of the promoter revealed that the key transcriptional region was located between -2128 to -1302. MAPPER predicted several potential transcriptional factors. Comparing the expression levels of these transcription factors in the adipose tissue of the obese and non-obese people, we found that NF-κB and FOXO1 are upregulated in obese people,implying that NF-κB and FOXO1 are the upstream repressor. Conclusion The promoter of human Disulfide-bond-A oxidoreductase-like Protein was successfully constructed, and its promoter activity was analyzed preliminary, which established the foundation for the further transcriptional regulation research.
keywords:Disulfide-bond-A oxidoreductase-like protein  Transcriptional regulation  Promoter
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