周锐;甘露;

• 1:四川大学华西医院生物治疗国家重点实验室心血管疾病研究室Ⅰ

摘要(Abstract)：

[目的]利用分子克隆技术,构建一套体外miRNA/mRNA-3′UTR interactions reporter system并鉴定其功能。[方法]分别以质粒pIRES2-EGFP、pGL3-Control为模板PCR扩增EGFP,Luciferase编码序列,并依次在5′,3′添加酶切位点XhoⅠ、HindⅢ,定向克隆到pBlue-script-KS(-)中的多克隆位点中,利用该载体中的多克隆位点序列使用XhoⅠ、NotⅠ酶切获取3′带有一段常用酶切位点的多克隆序列,最后定向克隆到pIRES2-EGFP中,由于取代了前者中的核糖体进入位点序列(IRES)及EGFP,而其载体骨架为pUC系列且克隆片段下游增加了一段包括HindⅢ、EcoRⅠ、BamHⅠ、XbaⅠ、NoⅠt等常用酶切位点的多克隆位点序列,为miRNA及mRNA-3′UTR的构建提供了便利的条件,故新构建载体分别取名为pUC-EGFP-MCS,pUC-Luciferase-MCS。构建pUC-EGFP-pre-miRNA1及pUC-Luciferase-HDAC4-3′UTR共转染HEK293-T细胞,使用荧光定量PCR检测miRNA1的过表达情况并测定相对荧光素酶活性,以检测所建立系统是否有效。[结果]所构建载体pUC-EGFP-MCS、pUC-Luciferase-MCS经酶切及测序证明构建正确;阳性对照组pUC-EGFP-pre-miRNA1及pUC-Luciferase-HDAC4-3′UTR共转染细胞后miRNA1得到有效表达且相对于对照组,即pUC-EGFP-MCS,pUC-Luciferase-HDAC4-3′UTR,试验组的相对荧光素酶活性显著降低。[结论]成功构建In-vitro miRNA/mRNA-3′UTR interactions reporter system,为体外筛选鉴定miRNA/mRNA-3′UTR interactions提供了一个有力的技术平台。

关键词(KeyWords)： 微RNA;信使RNA非翻译区;荧光素酶报告系统

Abstract:

Keywords:

基金项目(Foundation):

作者(Author): 周锐;甘露;

Email:

DOI: 10.13989/j.cnki.0517-6611.2009.22.031

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