靶向增强绿色荧光蛋白shRNA的真核表达载体构建  

Construction of Eukaryotic Expression Vector Containing shRNA Targeting EGFP

作　　者：包莉[1] 

机构地区：[1]襄樊职业技术学院南校区医学院,湖北襄樊441021

出　　处：《湖北民族学院学报（医学版）》2009年第2期4-6,F0003,共4页Journal of Hubei Minzu University(Medical Edition)

基　　金：湖北省教育厅科技研究计划中青年人才项目(Q20092904)

摘　　要：目的构建针对增强绿色荧光蛋白(EGFP)的shRNA真核表达载体pGenesil-1-EGFP-shRNA/U6。方法设计并合成针对EGFP的shRNA DNA片段,退火后连接到真核表达载体pGenesil-1上,通过测序证实载体构建成功;用脂质体将构建正确的质粒转染到中国仓鼠卵巢(CHO)细胞中,72 h后在荧光显微镜下观察EGFP的表达。结果测序证实针对EGFP的shRNA真核表达载体pGenesil-1-EGFP-shRNA/U6构建正确;荧光显微镜下观察转染pGenesil-1-EGFP-shRNA/U6质粒的CHO细胞中,荧光明显减少。结论针对EGFP的shRNA真核表达载体pGenesil-1-EGFP-shRNA/U6能明显干扰EGFP的表达。Objective To construct pGenesil - 1 - EGFP -shRNA/U6 eukaryotic expression vector containing double strands DNA for RNA interference on EGFP. Methods A recombinant,eukaryotic expression vector pGenesil - 1 - EGFP - shRNA/U6 was generated by cloning a double strands DNA for RNA interference on EGFP into pGenesil - 1 vector. After sequencing, the pGenesil- 1 - EGFP - shRNA/U6 plasmid was transfected into CHO cells by lipofectamineTM2000, after 72 hours, the EGFP expression was observed under fluorescent microscope. Results The pGenesil - 1 - EGFP - shRNA/U6 recombinant eukaryotic expression vector had been constructed correctly after sequencing. The fluorescence was reduced obviously in CHO cells transfected with pGenesil - 1 - EGFP - shRNA/U6 plasmid. Conclusion The pGenesil - 1 - EGFP- shRNA/U6 plasmid can knockdown the expression of EGFP obviously.

关 键 词：增强绿色荧光蛋白 小发夹RNA RNA干扰 真核表达载体 

分 类 号：R730.51[医药卫生—肿瘤]