Cell-SELEX技术筛选急性早幼粒细胞白血病NB4细胞适配体

目的用以细胞为靶标的指数富集配体的系统进化(Cell-SELEX)技术获得与急性早幼粒细胞白血病(APL)细胞株NB4高亲和力、高特异性的适配体。方法以NB4细胞为靶标,用Cell-SELEX技术从随机单链DNA(ssDNA)文库中筛选出一组适配体,将筛选得到的适配体群进行克隆、测序及结合力测定,用流式细胞仪和荧光显微镜对结合率最高的适配体进行亲和力和特异性分析。结果第1,4,7,10,13,16,19轮筛选获得的次级文库与NB4细胞的结合率分别为(1.6%,3.8%,6.3%,11.4%,15.4%,19.9%,16.7%);第16轮筛选的21个阳性克隆菌测序结果表明,存在有3种高度富集的适配体,分别为CX1序列2个,CX5序列3个,CX9序列16个;流式细胞仪检测3种序列的结合率分别为9.7%,12.6%,17.2%;CX9的解离常数(Kd)为16.2 nmol/L;荧光显微镜下发现,CX9与NB4细胞结合的荧光强度高于K562细胞。结论用Cell-SELEX技术成功筛选到针对NB4细胞的适配体CX9,为荧光标记适配体快速鉴定APL提供实验依据。

Selection of aptamers for acute promyelocytic leukemia NB4 cells with Cell-SELEX

Objective To obtain the aptamer targeted to acute promyelocytic leukemia(APL) NB4 cells with high affinity and specificity by using the cell based systematic evolution of ligands by exponential enrichment(Cell-SELEX).Methods A group of aptamers target to NB4 cells were selected from a random single-stranded DNA(ssDNA) library by using Cell-SELEX.The screened aptamers were further cloned,sequenced and the binding forces were determined.One of the screened aptamers with the highest binding rate was isolated and its affinity and specificity were analyzed with flow cytometry and fluorescence microscope.Results The binding rates between NB4 cells and the secondary libraries acquired in the 1st,4 th,7th,10th,13th,16th and 19th selection rounds were 1.6%,3.8%,6.3%,11.4%,15.4%,19.9% and 16.7%,respectively.The sequencing results of 21 positive clones selected from the 16th round indicated that 3 kinds of aptamers were highly enriched,including the sequences from 2 of CX1,3 of CX5 and 16 of CX9.The determination of flow cytometry showed that the binding rates of aptamers CX1,CX5 and CX9 were 9.7%,12.6% and 17.2%,respectively,and the equilibrium dissociation constant(Kd) of CX9 reached to 16.2 nmol/L.The fluorescence intensity of aptamer CX9 binding to NB4 cells was stronger than that of K562 cells under fluorescence microscope.Conclusion The CX9 aptamer with high affinity and specificity to NB4 cells was obtained by using Cell-SELEX successfully,which provides the evidence for the rapid identification of APL with the fluorescence-labeled aptamer.