人慢性粒细胞白血病融合蛋白BCR-ABL适配子筛选及结构分析

Screening and characterization of aptamers of human chronic myeloid leukemia( CML) fusion protein BCR-ABL and its structure analysis

目的:筛选、鉴定人慢性粒细胞白血病融合蛋白BCR-ABL适配子。方法:利用SELEX(Systematic evolution of ligands by exponential)技术,以高纯度融合蛋白BCR-ABL为靶分子,从体外化学合成的长度为90 bp的随机单链DNA文库中来筛选与融合蛋白BCR-ABL特异性结合的寡核苷酸适配子,并进行解离常数(Kd)值测定和适配子序列测定,再分别用Clustal W软件包和DNA Folding Sever分析适配子一级结构及二级结构,以酶联仪测定OD450值,根据OD值高低判定适配子亲和力大小。结果:经过13轮筛选,随机ss DNA文库与融合蛋白的亲和率从0.3%上升到47.1%,所有的一级结构没有共同的同源序列,二级结构分析结果显示,茎和环等二级结构可能是适配子和融合蛋白BCR-ABL结合的基础,其中,寡核苷酸适配子A2与BCR-ABL亲和力最高,kd值达72 nmol/L。结论:利用随机寡核苷酸文库筛选技术成功获得抗融合蛋白适配子,为临床上治疗和预防慢性粒细胞白血病提供一定的参考。

Objective： To screen and characterize aptamers against BCR-ABL fusion protein. Methods： A 90 bp single stranded DNA（ ss DNA） random library was subjected to 13 rounds of selection against BCR-ABL fusion protein by systematic evolution of ligands by expotential enrichment（ SELEX） method,the selected aptamers were cloned and sequenced. The primary sequences and structure of aptamers were analyzed by Clustal W and DNA Folding Sever and the percentage of the ss DNA pool bound to BCR-ABL core protein were determinated. Results： after 13 rounds selection,the percentage of ss DNA pool bound to BCR-ABL fusion protein increased from 0. 3% to 47. 1%,the results showed that affinities of the Aptamers were different,the second structure analysis revealed possible stem-loops for binding to BCR-ABL fusion protein,the affinity of aptamer A2 to BCR-ABL fusion protein was highest with Kd values as low as 72 nmol / L. Conclusion： Aptamers against BCR-ABL fusion protein has been identified by SELEX methods from a 90 bp single stranded DNA library. And provide certain reference for the clinical treatment of chronic myelogenous.