含Egr-1基因启动子的报告质粒的构建及Purα对Egr-1基因表达的调控  

Establishment of Reporter Plasmid Containing Egr-1 Promoter and Regulatory Effects of Purα on Egr-1 Gene Expression

作　　者：袁程敏 柴娟[3] 李平[2] 张冰莹[2] 郭姗姗[1] 何文欣[1] 石晓光[1] 孙涛[1] 崔建奇[1,2] 

机构地区：[1]宁夏医科大学颅脑疾病重点实验室,国家重点实验室培育基地,银川750004 [2]宁夏医科大学基础医学院生物化学与分子生物学系,银川750004 [3]浙江省金华市人民医院生殖中心,金华321000

出　　处：《宁夏医科大学学报》2017年第4期368-373,共6页Journal of Ningxia Medical University

基　　金：国家自然科学基金(81260197);宁夏脑计划(2016BZ07);宁夏医科大学优势学科群建设科研项目(XY201511)

摘　　要：目的探讨人转录活化因子阿尔法又称富含嘌呤成份(或元件)结合蛋白(human transcriptional activator alpha,also named as purine-rich element binding protein alpha,Purα)对早期生长反应蛋白-1(Egr-1)基因表达的调控作用。方法利用PCR技术从U87MG细胞基因组DNA中扩增Egr-1基因启动子近5’端约700bp的DNA片段,将扩增的DNA片段克隆到萤火虫荧光素酶报告基因载体p GL3-Basic的相应酶切位点,构建成含有Egr-1启动子的萤火虫荧光素酶报告基因载体(p GL3-Basic-Egr-1)并通过酶切和测序进行鉴定。通过萤火虫荧光素酶实验对p GL3-Basic-Egr-1启动子的活性进行测定并检测Purα蛋白对p GL3-Basic-Egr-1启动子活性的作用。通过q PCR和Western blot实验检测Purα蛋白在转录和翻译水平对Egr-1基因表达的调控作用。结果通过序列测定和酶切分析证实所构建的含Egr-1基因启动子的报告载体与实验设计完全一致,萤火虫荧光素酶分析实验证实所构建的p GL3-Basic-Egr-1启动子序列正确并具有活性,Purα蛋白可以抑制p GL3-Basic-Egr-1启动子的活性,过表达Purα可以在转录和翻译水平下调Egr-1基因的表达。结论 Purα蛋白具有下调Egr-1基因表达的作用。Objective To investigate the regulatory effects of human transcriptional activator Purα on early growth responsive protein-1（Egr-1）gene expression and to reveal the regulatory mechanism. Methods 700 bp DNA fragments of Egr-1 promoter in proximal end were amplified from the genomic DNA extracted from U87 MG cells and the amplified DNA fragments were inserted into the relevant cloning site of luciferase reporter plasmid p GL3-Basic to establish the luciferase reporter plasmid of Egr-1. The constructed plasmid was identified with endonuclease cutting and sequencing. The activity of Egr-1 promoter was tested with luciferase assay and the effects of Purα on p GL3-Basic-Egr-1 promoter was evaluated. The regulatory effects of Purα on p GL3-Basic-Egr-1 in transcriptional and translational levels were assessed with real time PCR and western blot assay. Results The constructed Egr-1 luciferase reporter plasmid was accordance with the experimental design when identified with endonuclease cutting and sequencing, luciferase assay confirmed that the constructed p GL3-Basic-Egr-1 reporter plasmid possesses correct DNA sequence and satisfied activity. Purα could down-regulate p GL3-Basic-Egr-1 promoter activities. Overexpression of Purα could repress Egr-1 gene expression both in transcriptional and translational levels. Conclusion Purα can down-regulate Egr-1 gene expression.

关 键 词：Purα 早期生长反应蛋白-1 基因表达调控 阿尔茨海默病 

分 类 号：R749.16[医药卫生—神经病学与精神病学]