靶向CXCR1基因的shRNA真核表达质粒的构建及鉴定  

Construction and identification of eukaryotic expression plasmid encoding shRNA against CXCR1 gene

作　　者：王俊普[1] 胡婉明[1] 王宽松[1] 肖燕[1] 李景和[1] 文继舫[1] 

机构地区：[1]中南大学基础医学院病理学系,湖南长沙410013

出　　处：《中国普通外科杂志》2013年第4期456-462,共7页China Journal of General Surgery

基　　金：湖南省研究生科研创新资助项目(CX2011B046);湖南省长沙市科技计划资助项目(K1005005-31;K1106041-31)

摘　　要：目的:构建靶向化学趋化因子受体1(CXCR1)的短发夹小干扰RNA(shRNA)质粒表达载体。方法:针对人CXCR1基因的mRNA序列,按RNA干扰靶位点的设计原则,设计并构建靶向CXCR1基因的3个shRNA质粒表达载体和1个阴性对照质粒表达载体,经酶切和测序确认构建成功后,转染胃癌细胞MKN45,RT-PCR和Western blot检测CXCR1 mRNA和蛋白的表达。结果:经酶切和测序证实,3个靶向CXCR1基因的shRNA真核表达质粒均构建成功;与未转染和转染阴性对照质粒的MKN45细胞比较,转染3种shRNA质粒的MKN45细胞,CXCR1 mRNA和蛋白水平均明显下调(均P<0.05)。结论:靶向CXCR1基因的shRNA真核表达质粒的成功构建,为进一步研究CXCR1在胃癌中的功能和实验性靶向治疗提供了初步的基础。Objective： To construct the plasmid expression vector that expresses short hairpin RNA （shRNA） against CXC chemokine receptor 1 （CXCR1）. Methods： Three shRNA expression vectors targeting CXCR1 gene and one negative control （non-targeting sequence） expression vector were designed and constructed according to the mRNA sequence of CXCR1 gene and RNA interference design guidelines. After identification with restriction enzyme digestion and sequencing analysis, these vectors were transfected into gastric cancer MKN45 cells in vitro, and then the CXCR1 mRNA and protein expressions in the cells were detected by RT-PCR and Western blot analysis.Results： As identified by enzyme digestion and sequencing analysis, all the three shRNA eukaryotic expression plasmid vectors targeting CXCR1 gene were successfully constructed. Compared with untransfected MKN45 cells or MKN45 cells transfected with negative control vector, both mRNA and protein levels of CXCR1 were significantly reduced in MKN45 cells transfected with any of the three shRNA vectors （all P〈0.05）. Conclusion： The successful construction ofplasmid expression vector encoding shRNA against CXCR1 may provide a preliminary step for further investigation of the role of CXCR1 in gastric cancer and experimental targeted therapy.

关 键 词：胃肿瘤 受体 CXCR RNA 小分子干扰 

分 类 号：R735.2[医药卫生—肿瘤]