随机单链DNA文库SELEX筛选寡核苷酸适配子方法的建立  

A Procedure for SELEX Screening Aptamers From ssDNA Random Library

作　　者：詹林盛[1] 邵宁生[2] 彭剑淳[1] 孙红琰[1] 王全立[1] 

机构地区：[1]军事医学科学院输血医学研究所,北京100850 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《生物化学与生物物理进展》2003年第1期151-155,共5页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(30200352);全军医药卫生科研基金(01NB061)资助项目。~~

摘　　要：指数富集配基的系统进化 (SELEX)技术是一种新的组合化学技术 .体外构建了一个长度为 81nt、含有35个随机序列的单链DNA (ssDNA)文库 ,优化了ssDNA文库扩增为双链DNA (dsDNA)文库的PCR反应条件 .通过对比不对称PCR和生物素 -链亲和素磁珠分离方法制备ssDNA文库的效果 ,确定了以生物素 -链亲和素磁珠分离方法制备ssDNA .由于脱氧核糖核酸的疏水性导致ssDNA文库与硝酸纤维素滤膜的结合背景过高 ,因此选择以微孔板为介质 ,分离与靶蛋白结合的适配子 .经过 9轮循环筛选 ,随机ssDNA文库与丙型肝炎病毒(HCV)核心蛋白 (C蛋白 )的结合率从 0 . 5 %上升到 32 . 5 % .SELEX (systematic evolution of ligands by exponential enrichment) is a new combinatorial chemistry technology. An 81 nucleotides ssDNA pool containing 35 random nucleotides flanked by invariant primer was designed. The PCR amplification conditions were optimized for converting the ssDNA pool into dsDNA pool. Compared to asymmetry PCR, the technique based on biotin-streptavidin-magnetism bead was suitable for generation of ssDNA from dsDNA. Since the hydrophobicity of deoxyribose leads to an inherently higher degree of background binding to nitrocellulose filters, panning procedures on microtiter plates were employed. After 9 rounds selection, the percentage of the ssDNA pool bound to HCV core protein inceased from 0.5% to 32.5%.

关 键 词：随机单链DNA文库 SELEX技术 筛选 寡核苷酸适配子 丙型肝炎病毒核心蛋白 

分 类 号：R392－33[医药卫生—免疫学] Q78[医药卫生—基础医学]