人Rac1基因shRNA表达质粒的构建及鉴定  

Construction and Identification of shRNA Expression Vector for Human Rac1 Gene

作　　者：王虹[1,2] 张菊青[1] 陈玉丙[1] 张红梅[3] 崔满华[1] 

机构地区：[1]吉林大学第二医院,长春130041 [2]北华大学附属医院妇产科,吉林吉林132011 [3]吉林大学中日联谊医院唐敖庆特聘教授实验室,长春130033

出　　处：《中国生物制品学杂志》2011年第4期414-417,共4页Chinese Journal of Biologicals

基　　金：吉林省自然科学基金项目(201015175)

摘　　要：目的构建人Rac1基因shRNA表达质粒,为提高卵巢癌放化疗的敏感性奠定基础。方法根据GenBank中登录的人Rac1基因序列,应用shRNA设计软件设计并合成用于构建shRNA表达质粒的oligo DNA,构建shRNA重组质粒。将阳性Rac1基因shRNA重组质粒经脂质体介导转染卵巢癌Skov3细胞,48 h后经RT-PCR筛选有效的shRNA重组质粒。结果经限制性内切酶BamHⅠ和PstⅠ酶切鉴定出阳性Rac1基因shRNA重组质粒,序列比对分析结果与理论序列完全一致。RT-PCR检测显示,转染pGPU6/GFP/Rac1-524的Skov3细胞Rac1基因mRNA的转录水平下降。结论已成功构建人Rac1基因shRNA表达质粒,并筛选出有效的干扰质粒pGPU6/GFP/Rac1-524。Objective To construct the shRNA expression vector for human Rac1 gene and lay a foundation of increasing the sensitivity of ovarian cancer to radiotherapy and chemotherapy.Methods The oligo DNA for construction of shRNA expression vector was designed and synthesized by using shRNA design software according to the sequence of human Rac1 gene in GenBank,based on which recombinant shRNA vector with human Rac1 gene was constructed and tranfected to ovarian cancer Skov3 cells,and effective recombinant shRNA plasmid was screened by RT-PCR 48 h later.Results Recombinant shRNA plasmid with Rac1 gene was identified by digestion with BamHⅠ and PstⅠ,of which the sequence was completely consistent with theoretical sequence.RT-PCR showed that the transcription level of Rac1 mRNA in Skov3 cells transfected with plasmid pGPU6/GFP/Rac1-524 decreased.Conclusion The shRNA expression vector for human Rac1 gene was successfully constructed,and effective interfering plasmid pGFU6/GFP/Rac1-524 was screened.

关 键 词：RAC1 GTP结合蛋白质 RNA干扰 卵巢肿瘤 

分 类 号：Q784[生物学—分子生物学]