利用绿色荧光蛋白进行microRNAs靶点分析的研究

目的利用绿色荧光蛋白作为报告基因,研究微RNAs(microRNAs)与靶点相互作用。方法在pEGFP-C1下游非编码区插入含miR-122a作用靶序列HCV 5′UTR,构建pEGFP-UTR载体,将miR-122a与pEGFP-UTR共转染HEK293细胞,观察增强绿色荧光蛋白EGFP表达强度变化,并与双荧光素酶报告系统比对。结果 miR-122a能明显抑制含UTR靶序列的EGFP蛋白表达,与双荧光素酶报告系统相比,绿色荧光蛋白在检测灵敏度上与双荧光素酶相接近。结论利用绿色荧光蛋白作为报告基因,能够清楚直观反映miRNAs与靶点作用情况。

Study of utilizing green fluorescent protein to analyze microRNAs targets

Objective To investage the interaction between microRNA and its target using enhanced green fluorecence protein(EGFP) as report gene.Methods MiR-122a target gene hepatitis C virus 5′untranslated region(UTR) was inserted into pEGFP-C1 to construct plasmid pEGFP-UTR.pEGFP-UTR and miR-122a were cotransfected into HEK293 cell line.At 48h after transfection,EGFP expression was detected by fluorecence microscope as well as Western blot.Meanwhile,the sensitivity of EGFP as report gene was evaluated by luciferase report gene.Results miR-122a could inhibit EGFP expression loading HCV UTR at its 3′ untranslated region.And the sensitivity of EGFP was close to luciferase.Conclusion EGFP can be used as report gene to reflect microRNAs and target interaction clearly and directly,and the feature of EGFP make it a good material for screening microRNAs and its unkonwn target.