检测miRNA活性的双荧光素酶单载体

A Dual Luciferase Single Plasmid to Monitor miRNA Activity

本研究报道了微小RNA靶分子鉴定及其活性分析的内参内置型双荧光素酶单载体与其应用.利用非连接酶依赖的基因克隆技术,借助一步式二元搭桥耦联长距离PCR及大肠杆菌体内同源重组方法,将萤火虫荧光素酶基因(Firefly luc)融合到pRL-TK载体的海肾荧光素酶基因(Renillaluc)和氨苄青霉素抗性基因之间,构建为两种荧光素酶基因表达框并置的单载体报告系统,命名为pMiSensor.在海肾荧光素酶基因的3'非翻译区引入多克隆位点Xba I和Apa I,便于克隆目的基因的3'UTR.海肾荧光素酶为报告基因,萤火虫荧光素酶为内参基因.多种哺乳动物细胞系的转染实验证实,pMiSensor可同时有效表达两种报告基因,其酶活显示出宽广的线性范围.通过构建pMiSensor-CCNE1报告载体,证明pMiSensor能够重现miR16对细胞周期蛋白CCNE1的调控作用.通过转染miR16抑制剂,证明pMiSensor-CCNE1可作为一种灵敏的生物感应器,探测细胞内微小RNA的活性变化.该双荧光素酶单载体具有重复性高、操作简便、定量准确的优点,适用于微小RNA靶分子的筛选、鉴定和确认,也适用于在细胞水平定量分析微小RNA的活性变化.

This study describes the construction and application of pMiSensor,a dual luciferase single plasmid system for miRNA target identification and functional analysis.Firefly luciferase was fused between Renilla luciferase and Ampicillin resistance genes in pRL-TK vector using ligase-independent cloning by bridge PCR and in vivo recombination in E.coli.Xba I and Apa I restriction sites were introduced into the 3′ UTR of Renilla luciferase for cloning of sequences to be tested.In this single plasmid,firefly luciferase serves reporter and Renilla luciferase works as internal control.pMiSensor transfection into cultured cells of multiple mammalian species in vitro allows the expression of the two luciferases simultaneously.pMiSensor-CCNE1 biosensor was able to verify the regulation of miR16 on CCNE1 and could also be used to report on the endogenous miR16 through the transfection of miR16 inhibitor with four types of trasnfection reagents.pMiSensor is a convenient and quantifiable tool that is applicable for miRNA target