慢性髓细胞性白血病K562细胞适体的筛选与鉴定
Screening and characterization of aptamers of chronic myelognous leukemia K562 cells
刘玲玲 1韩跃武 1祝凯华 2李真真 1韩亚萍 1路艳 1王春霞1
作者信息
- 1. 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
- 2. 兰州大学,第一医院普外二科,甘肃,兰州,730000
- 折叠
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CML K562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 V 2.5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.
Abstract
AIM: To screen and characterize oligonucleotide aptamers of chronic myelognous leukemia K562 cells. METHODS: Oligonucleotide aptamers specifically binding to chronic myelog-nous leukemia K562 cells were screened from 88 nt random ssDNA library in vitro syntbesis by SELEX method, sub-library was prepared by biotin-streptavidin magnetic beads and neutro-phils 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 random aelection were sequenced after screening by the blue and white. The affinities of the screened aptamers binding to chronic myelognous leukemia K562 cells were detected by fluorescent primers. Homology analyses of the primary structure and secondary structure prediction to the screened aptamters were conducted with Clustal 2.05 and DNA sis V 2.5 software. RESULTS: After 13 rounds of screening cycles, the absorbance (A) of the screened aptamers to chronic myelog-nous leukemia K562 cells rose from 0.12 up to 1.25 and the ahaorhance(A) of the screened aptamers did not significantly increase until the 13 th round of the screening. No homologous sequences were found by analysis of the primary structure but they could be divided into 6 families. Respective conserved series was found in 5 of the 6 families and no conserved series was found in only one family. Secondary structure analysis showed that the stem-loops and bulges structure of the aptamters might be the structure foundation of specific binding to chronic myelognous leu-kemia K562 cells. CONCLUSION: We have successfully screened out the oligonucleotide aptamers of high-affinity binding to chronic myelognous leukemia K562 cells by SELEX method.
关键词
白血病,髓样,慢性/K562细胞/SELEX/适体Key words
leukemia, myeloid, chronic/K562 cell/SELEX/aptamer引用本文复制引用
基金项目
国家高技术研究发展计划(863计划)(2006AA10A208)
甘肃省科技攻关项目(2GS064-A43-020-21)
出版年
2009
NETL
NSTL
维普
万方数据