诱导型eIF3g人工microRNA表达载体的构建及应用  

Construction and application of inducible artificial microRNA expression vector targeting eIF3g gene

作　　者：褚丽莎[1,2] 韩娜[2] 宋向阳[1] 曹江[2] 

机构地区：[1]浙江大学附属邵逸夫医院普外科,浙江杭州310016 [2]浙江大学邵逸夫临床医学研究所浙江省生物治疗重点实验室,浙江杭州310016

出　　处：《细胞与分子免疫学杂志》2011年第2期135-138,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30471955);浙江省自然科学基金资助(R206060)

摘　　要：目的:构建可用四环素调控的针对人eIF3g的人工microRNA表达载体,用于抑制肿瘤细胞中eIF3g的表达。方法:利用网络资源设计针对人eIF3g的人工microRNA,以PCR方法克隆,经DNA测序验证后亚克隆至四环素调控表达系统的pRevTRE2载体上,得到pRevTRE2-eIF3g-microRNA。将四环素调控表达系统的Tet-off质粒转染K562细胞获得稳定转染克隆后,再利用带有报告基因GFP的pRevTRE2-GFP转染选择调控能力较好的K562/Tet-off细胞株,将pRevTRE2-eIF3g-microRNA转染K562/Tet-off细胞,Westernblot检测eIF3g的表达,以研究eIF3g人工microRNA的作用效果。结果:DNA测序结果显示,针对人eIF3g的人工microRNA的克隆正确。pRevTRE2-GFP转染结果表明所选择的K562/Tet-off细胞株有较好的诱导功能。Westernblot结果显示,转染pRevTRE2-eIF3g-microRNA的K562/Tet-off细胞可用四环素调控eIF3g人工microRNA的表达从而抑制K562细胞中eIF3g的表达。结论:成功构建了可用四环素调控的eIF3g人工mi-croRNA表达载体,能有效调控K562细胞中eIF3g的表达。AIM： To construct an inducible artificial microRNA expression vector targeting elF3g gene and use it to inhibit the expression of eIF3g in K562 cells. METHODS： The microRNA targeting human elF3g was designed and obtained by PCR. After confirmed by sequencing, the microRNA was cloned into the pRevTRE2 plasmid. The Tet-off plasmids were transfected into K562 cells and selected for the stable tetracycline inducible K562/Tet-off transfectants. The pRevTRE2-eIF3g-miRNA plasmid was then transfected into stable K562/Tet-off transfectants and the expression of elF3g was detected by Western blot. RESULTS： The microRNA targeting elF3g was confirmed by sequencing. GFP fluorescence assay showed that the stable transfectants of Tet-off plasmids were under tetracycline control. Western blot results showed that the stable transfectants of pRevTRE2-eIF3g-microRNA inhibited elF3g expression under the regulation of tetracycline. CONCLUSION： The tetracycline-inducible artificial microRNA expression vector targeting eIF3g gene has been successfully constructed and effectively inhibits elF3g expression in K562 cells.

关 键 词：eIF3g MICRORNA 四环素诱导表达系统 

分 类 号：R733.7[医药卫生—肿瘤]