AFP基因SiRNA表达质粒的构建及鉴定  

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

作　　者：亓同钢[1] 汪运山[2] 王 芳[1] 吴文秀[1] 关广聚[1] 

机构地区：[1]山东大学第二医院临床分子生物学实验室,山东济南250000 [2]山东大学临床医学院济南市中心医院中心实验室,山东济南250000

出　　处：《山东大学学报（医学版）》2004年第3期353-354,358,共3页Journal of Shandong University:Health Sciences

基　　金：山东省自然科学基金资助课题(Z 2003 Col)

摘　　要：目的:构建针对人甲胎蛋白(AFP)基因siRNA表达质粒,为进一步研究AFP基因功能奠定基础?方法:选择RNA干涉靶序列,体外合成两段互补的寡核苷酸,通过与线性化的pSilencer3.0-H1连接?转化大肠杆菌,扩增?纯化得到所需质粒,通过琼脂糖凝胶电泳及基因测序鉴定其分子量及插入片段的序列?结果:纯化的质粒的分子量为2.8 Kb,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AFP基因的siRNAs表达质粒?Objective: To construct siRNAs expressing plasmid aimed at AFP gene and study the function of AFP with RNA interference technology. Methods: First, a target sequence in the middle of AFP gene was selected, then according to the sequence, two complementary 65 mer oligonucleotides were synthesized with 5' single-stranded over-hangs according to the kit manual, which was ligated with the linearized psilencer3.0-H1. The plasmid was transformed into DH5α bacteria to amplify and then purified. The purified plasmid was identified by gel electrophoresis and sequencing. Results: The results of gel electrophoresis and sequencing showed that the plasmid was about 2.8 kb which was identical with the positive control,and the sequence was identical with what we have inserted in, and there was no aberrations such as mutation, deletion, or insertion. Conclusion: The plasmid which can express siRNAs aimed at AFP gene has been constructed successfully. [

关 键 词：质粒 甲胎蛋白类 基因表达 

分 类 号：Q782[生物学—分子生物学]