重组质粒pEGFP-N1-Twist的构建、表达及其对SKOV3细胞克隆形成能力的影响  

Construction of recombinant pEGFP-N1-Twist plasmid and its stable expression in SKOV3 cells

作　　者：尉春艳[1] 张熙[2] 孙学军[3] 彭慧霞[1] 史玉霞[1] 王桂贤[1] 

机构地区：[1]西安交通大学医学院第二附属医院妇产科,陕西西安710061 [2]西安交通大学医学院第二附属医院神经外科,陕西西安710061 [3]西安交通大学医学院第一附属医院普外科,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2014年第4期447-450,共4页Journal of Xi’an Jiaotong University（Medical Sciences）

摘　　要：目的构建含有Twist基因的重组质粒pEGFP-N1-Twist,为进一步研究上皮性卵巢癌的发生及转移机制奠定基础。方法生物合成Twist cDNA基因序列,定向克隆至pEGFP-N1表达载体内,将pEGFP-N1-Twist转染入SKOV3细胞系,用Real-time PCR和Western blot检测Twist基因的表达情况;通过克隆形成实验检测pEGFP-N1-Twist对SKOV3细胞的作用。结果 Twist基因的cDNA已克隆到真核细胞表达载体pEGFP-N1中,将pEGFP-N1-Twist转染入SKOV3细胞后,Twist基因/蛋白的表达明显上调,转染组较对照组克隆形成明显增加(P<0.05)。结论成功构建了Twist基因的真核表达载体并能在细胞内稳定表达,Twist基因能增强SKOV3细胞的克隆形成能力。Objective To construct eukaryotic expression vector of pEGFP-N1-Twist for future research on the occurence and metastasis of epithelial ovary cancer.Methods The cDNA sequence of Twist gene was synthetized and cloned into the plasmid of pEGFP-N1.The expression of Twist gene was detected by real-time PCR and Western blot.The effect of Twist on SKOV3 cells was examined by clone formation test.Results The cDNA fragment of Twist gene was cloned into pEGFP-N1.The recombinant eukaryotic expression vector of pEGFP-N1-Twist was constructed successfully.The SKOV3 cells transfected with pEGFP-N1-Twist plasmid expressed a high level of Twist protein.The colony-forming capability of SKOV3 cells transfected by pEGFP-N1-Twist increased significantly compared with the control group （P＜0.05）.Conclusion The recombinant plasmid pEGFP-N1-Twist has been constructed successfully.Twist gene can improve the colony-forming ability of SKOV3 cells.

关 键 词：重组质粒 TWIST基因 卵巢癌 真核表达载体pEGFP-N1 基因克隆 

分 类 号：R711.75[医药卫生—妇产科学]