| 慢性髓细胞性白血病K562细胞适体的筛选与鉴定 |
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| 引用本文： | 刘玲玲,韩跃武,祝凯华,李真真,韩亚萍,路艳,王春霞.慢性髓细胞性白血病K562细胞适体的筛选与鉴定[J].第四军医大学学报,2009,30(13):1157-1160. |
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| 作者姓名： | 刘玲玲 韩跃武 祝凯华 李真真 韩亚萍 路艳 王春霞 |
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| 作者单位： | 兰州大学,基础医学院生物化学与分子生物学研究所,甘肃,兰州,730000;兰州大学,第一医院普外二科,甘肃,兰州,730000 |
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| 基金项目： | 国家“863”计划重大课题项目(2006AA10A208); 甘肃省科技攻关项目(2GS064-A43-020-21) |
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| 摘 要： | 目的：筛选并鉴定出慢性髓细胞性白血病（CML）K562细胞的寡核苷酸适体.方法：体外合成长度为88个碱基的随机单链DNA（ssDNA）文库,采用生物素-链霉亲和素磁珠法制备次级文库,以正常人血液中提取的中性粒细胞为反筛细胞,利用指数富集的配基系统进化（SELEX）技术筛选出与CMLK562细胞特异结合的适体.将筛选得到的适体回收纯化后连接pGEM-T质粒载体,经蓝白筛选后,随机挑选24个克隆子进行序列测定.采用荧光标记引物法检测ssDNA文库与K562细胞的亲和力,并用Clustal 2.05和DNA sis V2.5软件对适体序列进行一级结构同源性分析和二级结构预测.结果：经过13轮循环筛选,CML K562细胞适体的A值从0.12上升到1.25,至第13轮A值无明显增高.一级结构分析无同源序列,但可分为6个家族,其中5个家族各自具有保守序列,家族6无保守序列.二级结构分析表明,适体形成的茎环、凸环结构可能是与K562细胞特异性结合的结构基础.结论：利用SELEX技术成功筛选出高亲和性的CML K562细胞适体.
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| 关 键 词： | 白血病 髓样 慢性 K562细胞 SELEX 适体 |
Screening and characterization of aptamers of chronic myelognous leukemia K562 cells |
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| LIU Ling-Ling,HAN Yue-Wu,ZHU Kai-Hua,LI Zhen-Zhen,HAN Ya-Ping,LU Yan,WANG Chun-Xia.Screening and characterization of aptamers of chronic myelognous leukemia K562 cells[J].Journal of the Fourth Military Medical University,2009,30(13):1157-1160. |
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| Authors: | LIU Ling-Ling HAN Yue-Wu ZHU Kai-Hua LI Zhen-Zhen HAN Ya-Ping LU Yan WANG Chun-Xia |
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| Institution: | LIU Ling-Ling1,HAN Yue-Wu1,ZHU Kai-Hua2,LI Zhen-Zhen1,HAN Ya-Ping1,LU Yan1,WANG Chun-Xia11Department of Biochemistry and Molecular Biology,Basic Medical College,2Second Department of General Surgery,First Hospital,Lanzhou University,Lanzhou 730000,China |
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| Abstract: | AIM:To screen and characterize oligonucleotide aptamers of chronic myelognous leukemia K562 cells. METHODS: Oligonucleotide aptamers specifically binding to chronic myelognous leukemia K562 cells were screened from 88 nt random ssDNA library in vitro synthesis by SELEX method, sub-library was prepared by biotin-streptavidin magnetic beads and neutrophils from blood of normal humans were used as anti-sieve cells. The screened aptamters were purified and connected to pGEM-T plasmid vector and 24 clones of ran... |
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| Keywords: | leukemia myeloid chronic K562 cell SELEX aptamer |
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