小鼠DUSP13＇-UTR双荧光素酶报告基因表达系统的建立及功能鉴定  

Construction of mouse DUSP1 3′-UTR dual luciferase reporter system and its function identification

作　　者：温晓梨[1] 孙宏卫[1] 欧小利[1] 王妮[1] 梅柱中[1] 姜勇[1] 

机构地区：[1]广州南方医科大学病理生理教研室、广东省蛋白质组学重点实验室,510515

出　　处：《解放军医学杂志》2013年第4期265-268,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81070955,81030055)~~

摘　　要：目的建立小鼠双特异性磷酸酶1(DUSP1)3'端非翻译区(UTR)双荧光素酶报告基因表达系统,并对其进行功能鉴定。方法提取RAW264.7细胞总RNA并反转录为cDNA,以其为模板通过PCR扩增获得小鼠DUSP1 3'-UTR全长序列,酶切后克隆至pGL3-control载体荧光素酶编码基因的下游,获得重组载体LuDP,经PCR、限制性内切酶酶切、DNA序列分析鉴定正确后与内参照质粒pRL-TK共转染NIH3T3细胞,48h后检测双荧光素酶报告基因的表达。将含有小鼠DUSP1 3'-UTR的荧光素酶表达模块与来源于pRL-TK质粒的RL表达模块克隆至pLenti6-TR质粒中,获得双荧光素酶报告基因表达载体LuDP/RL,将其与RNA结合蛋白HuR表达载体(pcDNA3.1-HuR-FLAG)、mmu-miR-101a分别共转染NIH3T3细胞,检测双荧光素酶的活性,分析HuR、mmu-miR-101对荧光素酶基因表达活性的影响。结果成功构建了双荧光素酶报告基因表达载体。小鼠DUSP1 mRNA 3'-UTR可显著下调与其相连的荧光素酶的表达活性(0.14±0.01倍,P<0.01);共转染HuR可上调荧光素酶的表达活性(1.40±0.20倍,P<0.05),共转染mmu-miR-101a则可下调荧光素酶表达活性(0.57±0.18倍,P<0.05)。结论成功构建了DUSP1 3'-UTR双荧光素酶报告基因表达系统,该系统可用于转录后调控元件对小鼠DUSP1基因表达的调控机制研究。Objective To construct mouse dual specificity phosphatase-1 （DUSP1） 3＇-untranslated region （3＇-UTR） dual luciferase reporter system, and to identify its function. Methods The total RNA of RAW264.7 murine macrophage cells was extracted and reverse-transcribed into cDNA. The full length of mouse DUSP1 3＇-UTR fragment was amplified by PCR and sub-cloned to the immediate downstream of luciferase cDNA in pGL3-control luciferase expression plasmid to obtain the recombinant plasmid pGL3-Luc-DUSP 1- 3＇UTR （LuDP）. LuDP was then verified by PCR, restriction endonuclease and DNA sequencing analysis. Plasmid LuDP was transiently co-transfected into NIH3T3 cells with internal control plasmid pRL-TK, and the effects of DUSP1 3＇-UTR on the expression of attached luciferase gene was analyzed with dual luciferase reporter assay system 48 hours after transfection. Both the lucfferase expression module from LuDP and renilla luciferase expression module from pRL-TK were co-subcloned into pLenti6-TR vector to construct dual luciferase reporter plasmid LuDP/RL. ~e recombinant plasmid LuDP/RL was co-transfected with pcDNA3.1-HuR-FLAG （the eukaryotic expression plasmid of binding protein HuR） and miRNA mimics mmu-miR-101a into NIH3T3 cells respectively. The effects of HuR and mmu-mi-R101a on the expression ofDUSP1 3＇-UTR attached luciferase gene were also analyzed with dual luciferase reporter assay system. Results The dual luciferase reporter plasmid LuDP/RL was successfully constructed. The mouse DUSP 1 mRNA 3＇-UTR significantly down-regulated the expression of attached luciferase gene in NIH3T3 cells （0.14± 0.01 fold, P〈0.01）. The co-transfected HuR up-regulated the expression of luciferase in LuDP/RL plasmid （1.40 ± 0.20 fold, P〈0.05）, and co-transfected mmu-miR-101a down-regulated the expression of LuDP/RL（0.57 ± 0.18 fold, P〈0.05）. Conclusion 3he constructed dual luciferase reporter plasmid LuDP/RL can be used to analyze the regulatory mechanism of mouse DUSP 1 gene expression con

关 键 词：荧光素酶类 基因 报告 转录 遗传 

分 类 号：R349.83[医药卫生—基础医学]