MeCP2特异性shRNA真核细胞表达质粒的构建与鉴定  

Construction and identification of the eukaryotic expression plasmid for MeCP2 specific shRNA.

作　　者：邱小华[1] 胡金霞[1] 柯爱武[1] 李羲[1] 

机构地区：[1]江西医学院第二附属医院分子中心,南昌330006

出　　处：《江西医学检验》2004年第6期495-497,共3页Jiangxi Journal of Medical Laboratory Sciences

摘　　要：目的构建MeCP2特异性SiRNA表达质粒pSilence-MeCP2。方法根据人MeCP2mRNA序列设计作用靶序列,化学合成两条互补的DNA寡核苷酸链,该序列中包含靶序列正义链、反义链,两者间的连接序列及与质粒相匹配的酶切位点序列,连接质粒后转化感受态细胞(E.Coli),使其在大肠杆菌内大量复制,提取质粒,PCR鉴定。结果合成的寡核苷酸序列与要求的序列完全相符,且以正确的方向插入。结论成功构建两个MeCP2特异性SiRNA表达质粒psilence-MeCP2/Ⅰ和psilence-MeCP2/Ⅱ。Objective To construct and identify the eukaryotic expression plasmid psilencer-MeCP2. Methods According to the Homo sapiens methy1-CpG binding protein 2(MeCP2) mRNA sequence, four strands of oligonucletide were designed and chemically syntheized, then annealed into two double strands in vitro. The two DNA strands were ligated with plasmid vector respectively, named psilencer-MeCP2/Ⅰ and psilencer-MeCP2/Ⅱ. These plasmaids were transformed into compenent cells(E.coli JM 109). After selective culture, recombined plasmids were extracted from E.coli, sequence was used to make sure that the two strands were inserted into plasmaids in correct sequence and direction respectively. Results The synthesized strands of oligonucletide contained correct and complete sequence of the shRNA, the MeCP2 specific shRNA has been inserted into eukaryotic expression. Conclusion The eukaryotic expression plasmid psilencer-MeCP2 has been constructed successfully.

关 键 词：甲基化CPG结合蛋白2 短发夹核糖核酸 抑癌基因 基因表达沉默 真核细胞 

分 类 号：R730.3[医药卫生—肿瘤]