利用果蝇细胞研究基因表达转录后调控的双报告基因系统的建立  

Construction of a Dual Reporter Gene System Detecting Gene Expression Controlled by Posttranscriptional Regulatory Elements in Drosophila Cells

作　　者：张婷[1] 肖强海[1] 韦有恒[1] 刘勇[1] 马维骏[1] 

机构地区：[1]上海交通大学医学院／中国科学院上海生命科学研究院健康科学研究所,上海200025

出　　处：《现代检验医学杂志》2010年第4期22-24,27,共4页Journal of Modern Laboratory Medicine

摘　　要：目的 构建能够在果蝇S2＊细胞中检测转录后调控元件对基因表达调控影响的双报告基因系统.方法 首先利用pAc5.1/V5-His c,pGL3-basic和pRL-SV40载体,构建能在果蝇体系中表达萤火虫荧光素酶的报告基因质粒pAc-Fluc,以及用作内参照表达海洋腔肠荧光素酶的质粒pAc-Rluc.将TNF-α基因mRNA的3＇UTR连接至pAc-Fluc的Luc编码区下游构建pAc-Fluc-TNF-α质粒,并与pAc-Rluc共转至果蝇S2＊细胞,检测两种荧光素酶的相对活性.结果 成功构建了能够在果蝇S2＊细胞中组成型表达的双报告基因系统;在TNF-α mRNA 3＇UTR控制下表达的荧光素酶活性比对照有显著下降.结论 可以利用该双报告基因系统在果蝇细胞中研究转录后调控元件对基因的表达调控.Objective To construct a dual reporter gene system to detect the gene expression controlled by posttran-criptional regulatory elements in Drosophila S2＊ cells. Methods The firefly lueiferase （Fluc） gene from pGL-3 basic was sub-cloned into pAcS. 1/V5-His c vector to construct pAc-Fluc plasmid. The Fluc coding sequence was replaced by renilla luciferase （Rluc） gene from pRL-SV40 for pAc-Rluc plasmid construction. The 3＇ UTR of TNF-α was PCR amplified and subcloned into the pAc-Fluc,located down-stream of the Fluc coding sequence to generate pAc-Fluc-TNF-a plasmid. The activities of luciferase were measured after co-transfection with pAc-Rluc into S2＊ cells. Results The recombinant plasmids had been successfully constructed in S2＊ cells. The activity of luciferase under control of TNF-α 3＇UTR was downregulated significantly compared to its control. Conclusion The constructed dual reporter gene system could be used to detect the gene expression controlled by posttranscriptional regulatory elements in drosophila cells.

关 键 词：转录后调控元件 双报告基因 荧光素酶 果蝇S2＊细胞 

分 类 号：Q786[生物学—分子生物学]