FOXP3基因3’-UTR段双荧光素酶报告载体的构建及其活性鉴定  

CONSTRUCTION OF3'-UTR DUAL LUCIFERASE REPORTER VECTOR OF FOXP3 AND EVALUATION OF ITS ACTIVITY

作　　者：陈泽志[1] 李劲频[2] 杜伟伟[2] 刘竞丽[2] 莫雪安[2] 

机构地区：[1]广西医科大学 [2]广西医科大学第一附属医院神经内科,南宁530021

出　　处：《广西医科大学学报》2014年第2期170-173,共4页Journal of Guangxi Medical University

基　　金：广西自然科学基金资助项目(No.桂科青0728071);广西卫生厅科研资助项目(No.Z2007084)

摘　　要：目的:构建FOXP3基因3’-UTR双荧光素酶基因报告载体,并通过检测荧光素酶活性,初步分析可能调控FOXP3基因表达的miRNAs。方法:利用PCR方法,根据FOXP3基因3’-UTR序列信息设计其扩增引物,以293T细胞基因组DNA为模板PCR扩增FOXP3基因的3’-UTR序列,将其克隆到pmiRB-REPORT双荧光素酶报告载体中;运用Targetscan软件预测可能与FOXP3基因3’-UTR相互作用的miRNAs;利用LipofectaminTM2000试剂将miRNAs mimics与构建好的FOXP3基因3’-UTR段双荧光素酶报告载体共转染于常规培养的293T细胞中,然后使用双荧光素酶试剂盒检测荧光素酶活性,并与空白对照相比较。结果:经酶切及基因测序验证,成功构建含有FOXP3基因3’-UTR段双荧光素酶基因报告载体;Targetscan软件预测显示,FOXP3基因3’-UTR可能是miR-608的作用靶位点;双荧光报告显示,miR-608mimics组比空白对照组[(0.700 0±0.016 41)比(1.000±0.055 75)(P<0.001)]FOXP3基因3’-UTR双荧光素酶基因报告载体的荧光素酶活性下降30%。结论:FOXP3基因3’-UTR段双荧光素酶基因报告载体构建成功,初步证实miR-608对FOXP3有调控作用。Objective：To construct the dual luciferase reporter assay vector which contains FOXP3 gene 3＇-untranslated region and analyze the miRNAs modulated FOXP3 expression by detecting the luciferase activity of FOXP3 gene 3＇-untranslated region.Methods：The 3＇-TTR fragment of FOXP3 gene was amplified by PCR from genomic DNA of 293T cells and inserted into a dual luciferase reporter vector （pmiR-RB-RE-PORTTM vector） which was digested by enzyme.The miRNAs targeting FOXP3 gene 3＇-untranslated region was predicted by target scan.293T cells were treated with the dual luciferase reporter assay vector or empty vector and miR-608 mimics or control through transfection reagent.The activity of luciferase was analysed by the Dual Luciferase Reporter Assay System.Results：3＇-UTR fragment of FOXP3 gene was successfully cloned into the pmiR-RB-REPORTTM vector,which was verified by XhoI digestion and DNA sequencing.The predicted miRNAs targeting 3＇-UTR of FOXP3 may be miR-608.As compared with the control group （1.000±0.055 75）,the luciferase activity of pmiR RB REPORTTM FOXP3 gene 3＇-UTR treated with miR-608 mimics （0.700 0±0.016 41） was decreased to 30% （P〈0.001）.Conclusion：The FOXP3 gene 3＇-UTR luciferase reporter vector was constructed successfully,and preliminary evidence showed that the expression of FOXP3 gene was modulated by miR 608.

关 键 词：双荧光素酶基因报告 转录因子叉头样p3 微小RNA-608 3'-非编码区 

分 类 号：R-33[医药卫生]