AFP基因特异性miRNA表达质粒的构建及鉴定  

Construction and identification of a plasmid expressing miRNA in mammalian cells aimed at AFP gene

作　　者：张玉玲[1] 栾英姿[2] 苏占涛[1] 郑燕[2] 郏雁飞[2] 汪运山[2] 

机构地区：[1]山东省医学科学院基础医学研究所 [2]济南市中心医院

出　　处：《山东医药》2008年第11期33-35,共3页Shandong Medical Journal

基　　金：山东省自然科学基金资助项目(Z2003C01)

摘　　要：目的构建针对人甲胎蛋白(AFP)基因的miRNA表达质粒,为进一步研究AFP基因功能奠定基础。方法沿mRNA序列寻找连续两个AA及后面的19个核苷酸,通过同源性比较,选择与其他任何基因无同源性的序列即作为潜在的miRNA靶位点,针对AFP基因的编码序列体外合成两段互补的寡核苷酸,与线性化的pcDNATM6.2-GW/EmGFP-miR RNAi EXPRESSION VECTOR载体连接后转化大肠杆菌,扩增、纯化获得所需质粒,以琼脂糖凝胶电泳、PCR及基因测序鉴定其相对分子质量及插入片段的序列。结果纯化质粒的相对分子质量为5.8 kb,PCR鉴定结果符合目的条带(260 bp左右)大小,插入的寡核苷酸序列与设计的序列完全相符。结论成功构建了针对人AFP基因的miRNA表达质粒。Objective To construct a AFP miRNAs expressing plasmid for function study of AFP gene with RNA interference technology. Methods A target sequence of AFP mRNA was selected; two complementary 64 mer oligonucleotides were synthesized according to target sequence, then ligated with linearized pcDNA^TM6. 2-GW/EmGFP-miR RNAi EXPRESSION VECTOR. The plasmid was transformed into One Shot TOP 10 bacteria; after amplification and then purification, it was identified by gel electrophoresis, PCR and sequencing. Results The constructed plasmid was about 5. 8 kb long, and the inserted soquence was identical with design, with no aberrations such as mutation, deletion, or insertion. Conclusion The AFP miRNA expressing plasmid can be successfully constructed.

关 键 词：质粒 甲胎蛋白 基因 表达 

分 类 号：Q782[生物学—分子生物学] R318[医药卫生—生物医学工程]