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.
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 aptamtcrs were purified and connected to pGEM-T plasmid vector and 24 clones of random selection 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 myelognous leukemia K562 cells rose from 0. 12 up to 1.25 and the absorbance （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 leukemia K562 cells. CONCLUSION： We have successfully screened out the oligonucleotide aptamers of high-affinity binding to chronic myelognous leukemia K562 cells by SELEX method.
出处
《第四军医大学学报》
北大核心
2009年第13期1157-1160,共4页
Journal of the Fourth Military Medical University
基金
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)
