pEGFP-N1-Fcy::Fur重组质粒的构建及其在卵巢癌细胞中的表达  

Construction of pEGFP-N1-Fcy::Fur recombinant plasmid and observation of its expression in ovarian cancer cells

作　　者：伍霞[1] 钟玲[1] 池余刚[1] 蒋兴伟[1] 王勇[1] 

机构地区：[1]重庆医科大学附属第二医院妇产科,重庆400010

出　　处：《重庆医科大学学报》2008年第11期1330-1333,共4页Journal of Chongqing Medical University

基　　金：重庆市卫生局课题(05-2-002)

摘　　要：目的:构建携带融合型自杀基因Fcy::Fur的荧光真核表达质粒pEGFP-N1-Fcy::Fur,并观察其在卵巢癌细胞中的表达。方法:应用基因重组技术,将pORF-Fcy::Fur中的Fcy::Fur目的基因亚克隆到荧光真核表达载体pEGFP-N1,以酶切1和测序鉴定重组质粒的正确性。应用脂质体介导的转染技术将该质粒导入SKOV3细胞,24h后观察绿色荧光蛋白(Green fluorescent protein,GFP)瞬时表达情况,用Westernblot方法检测Fcy::Fur表达。结果:酶切和测序鉴定证实插入片段正确。细胞转染24h后,荧光显微镜下观察到GFP表达,60%转染细胞发出绿色荧光。Western-blot检测到Fcy::Fur表达。结论:成功构建pEGFP-N1-Fcy::Fur荧光真核表达质粒,并可在卵巢癌细胞中有效表达,为卵巢癌基因治疗提供实验基础。Objective：To construct a EGFP （Enhanced green fluorescent protein）-labled euk-aryotic expression plasmid of Fcy：：Fur suicide gene and to detect its expression in SKOV3 cell line. Methods：With the technology of gene re-arrangement,Fcy：：Fur gene in pORF-Fcy：Fur plasmid was subcloned into pEGFP-N1 vector, with its correctness evaluated by the means of r-estriction enzyme analysis and sequencing.It was transfected into SKOV3 cells with lipofectin,the transient expression of GFP was observed under flu- orescence microscope after 24 hours and detected by Western blot. Results：Correct construction of pEGFP-N1-Fcy：：Fur was identified by methods of restriction enzyme analysis and nucleotide sequence determination.A total of 60% transfe-cted cells emitted out green fluorescence under flnorescent microscope after 24 h after transfecti-.on. Fcy：：Fur gene expressed by the transfected cells were testified by Western blot. Conclusion：The recombinant eukaryotic expression vectors have been constructed successfully and effective-ly expressed in ovarian cancer cells,which may provide an experimental basis for gene therapy of ovarian cancer.

关 键 词：自杀基因 Fcy::Fur 绿色荧光蛋白 基因转染 真核表达载体 

分 类 号：Q782[生物学—分子生物学] R322.6[医药卫生—人体解剖和组织胚胎学]