SELEX实验中核酸-蛋白复合物分离方法对富集库亲和力的影响  

Effect of different isolations for nucleic acid and protein complex on affinity of enriched library in SELEX experiment

作　　者：胡承香[1] 顾长国[1] 朱旭东[1] 李磊[1] 

机构地区：[1]第三军医大学附属大坪医院野战外科研究所第一研究室,重庆400042

出　　处：《第三军医大学学报》2003年第8期680-682,共3页Journal of Third Military Medical University

基　　金：国家重点基础研究发展规划资助项目 ("973"项目 )

摘　　要：目的　比较SELEX实验中 ,核酸 蛋白复合物分离技术的差异对筛选富集库亲和力的影响。方法　以 β 转化生长因子Ⅱ型受体为靶目标 ,采用不同技术路线从ssDNA随机库中获取亲和富集库 :技术路线 1,靶目标预先被吸附到 96孔微量滴定板后 ,再进行筛选 ,从滴定板上洗脱结合核酸 ;技术路线 2 ,溶液中筛选、硝酸纤维膜截留核酸 蛋白复合物 ,从膜上回收结合核酸 ;通过膜结合测定、凝胶阻滞测定实验检测筛选富集库对靶目标的亲和力。结果　经 4轮筛选后 ,技术路线 1来源的富集库与初始随机库相似 ,对靶目标无亲和力 ;而技术路线 2来源富集库对靶目标的亲和力较初始随机库有显著提高。结论　SELEX实验中 ,溶液中筛选、膜截留结合核酸的技术路线与固 液相筛选、固相中直接获取结合核酸的途径相比较 。Objective To compare the effect of different partition of nucleic acid protein complex on the affinity of enriched library in systematic evolution of ligands by exponential enrichment (SELEX). Methods Two protocols were adopted to select the enriched library to transforming growth factor β receptor Ⅱ(TGF β RⅡ). Protocol 1: protein was absorbed on the surface of 96 well plate; then, selection was carried out; the binding nucleotide acids were eluted from the supporter directly. Protocol 2: selection was done in solution; nucleotide acid protein complex was captured in nitrocellulose membrane; the binding nucleotide acids were obtained from membrane. Filter biding assay and gel shift assay were performed to detect the affinity of the enriched ssDNA library from different protocols. Results After 4 rounds of selection, the affinity to TGF β RⅡ was obviously improved in the enriched library from protocol 2 compared with the initial library, while no such improvement was found in the enriched library from protocol 1. Conclusion In the SELEX experiment, the way of selection in solution, then partition of the binding nucleotide acids in filter is easier to enrich the binding fragment from initial ssDNA random library, compared with the way of fixation of target protein to solid supporter, then selection between the solid phase and liquid phase and elution of the binding nucleotide acids from supporter.

关 键 词：SELEX APTAMER 富集库 亲和力 

分 类 号：R394－33[医药卫生—医学遗传学] R394.3[医药卫生—基础医学]