B淋巴细胞活化因子特异的单链DNA适体的人工进化

Selection of single-strand DNA aptamer specifically binding to B cell activating factor

目的:进化筛选与B淋巴细胞活化因子(B cell activating factor be longing to the TNF family,BAFF)特异结合的单链DNA适体,鉴定其结合力。方法:应用指数方式富集的配体系统进化(systemati cevolution of ligands by exponential enrich-ment,SELEX)技术,将随机序列寡核苷酸文库与BAFF分子相互作用,分离出结合的寡核苷酸配体,经反复扩增、筛选数个循环,获得重组人BAFF的单链DNA适体,应用Dot blotting鉴定其结合力,酶联法比较各适体序列对BAFF的相对结合力。结果:优化实验条件后成功进行了13轮次筛选,随机选取42个阳性克隆测序,各序列GC含量最高达75%,分为6类不同序列;Dotblotting证实这些适体均与BAFF相结合;比较各适体序列结合BAFF的光密度值,以序列44光密度值最高(0.752,P<0.05),具有对BAFF相对最高结合力。结论:首次成功获得特异结合BAFF的高亲和力的单链DNA适体,为研发防治肿瘤和自身免疫疾病新药奠定了基础。

Objective： To screen for single-strand DNA aptamers specifically binding B cell activating factor belonging to the TNF family（BAFF） and to identify its affinity. Methods： Systematic evolution of ligands by exponential enrichment （SELEX） technique was used in our study. The ssDNA ligand was separated by interaction between random oligonucleotide library and BAFF, then ssDNA aptamers binding recombinant human BAFF were obtained through PCR amplification. The binding ability of the aptamers was identified by dot blotting and the relevant affinities of each aptamer to BAFF were compared by enzyme linked aptamer assay. Results： Thirteen rounds of screening was performed under optimized condition. Six different sequences were obtained from 42 randomly-chosen positive clones, whose top GC content was up to 75%. Dot blotting confirmed that all the 6 sequences were suitable to bind with BAFF. Sequence 44 had the highest optical density in the 6 sequences in enzyme linked aptamer assay （P 〈 0.05 ）, indicating it has the highest bonding force to BAF. Condusion： The ssDNA aptamers binding BAFF has been obtained for the first time, which lays a base for developing new drugs for treating autoimmune diseases and tumors.