大鼠成骨细胞特异单链DNA适配体的筛选与鉴定  

Selection and Identification of ssDNA Aptamers Specific to Rat ROS1728 Cells

作　　者：李慧[1] 丁红梅[1] 李洁[1] 黄皑雪[1] 苏雪婷[1] 梁超[2] 张令强[2] 赵强[1] 李少华[1] 邵宁生[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《生物技术通讯》2015年第1期81-84,共4页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2012AA022501)

摘　　要：目的:筛选能特异识别大鼠成骨细胞的单链DNA(ssDNA)适配体并对其进行鉴定。方法:利用完整细胞为靶标的消减细胞SELEX技术筛选大鼠成骨细胞特异ssDNA适配体,通过荧光显微镜、流式细胞术、基因克隆测序、MEME在线软件和RNA structure分析软件,分析适配体的一、二级结构,并对筛选得到的适配体进行鉴定。结果:经过6轮消减细胞SELEX筛选,荧光显微镜鉴定文库已富集;通过流式细胞术检测及测序分析,得到2条适配体L54和L66与大鼠成骨细胞特异结合,其平衡解离常数分别为494.4±133.3和511.4±160.7 nmol/L。结论:筛选获得特异识别大鼠成骨细胞的ssDNA适配体。Objective： To select and identify ssDNA aptamers specific binding to rat ROS1728 cells. Methods： Subtractive SELEX technology targeting the whole intact cells was used to screen ssDNA aptamers which were specific binding to ROS1728 cells. The aptamers were identified by fluorescence microscoping, flow cytometry, gene cloning and sequencing. MEME online software and RNA structure analysis software were employed to predict the secondary structures of the aptamers. Results： The signal of fluorescence microscope showed sufficient pool enrichment after 6 rounds of subtractive SELEX. Flow cytometry results showed that aptamers L54 and L66 were capable of binding specifically to ROS1728 ceils but not to UMR106 cells and ICE-6 cells, with a dissociation constant of 494.4±133.3 and 511.4±160.7 nmol/L respectively. Conclusion： We have obtained the ssDNA aptamers specific binding to the ROS1928 ceils.

关 键 词：大鼠成骨细胞 DNA文库 适配体 SELEX筛选 鉴定 

分 类 号：Q781[生物学—分子生物学] Q25