筛选环孢霉素A适体的SELEX技术的建立

Establishment of SELEX technique for screening DNA aptamer for cyclosporina A

目的:运用指数富集的配体系统进化(SELEX)技术,筛选并鉴定环孢霉素A(CsA)特异性的适体。方法:合成一个全长78个核苷酸中间含35个随机序列的随机单链寡核苷酸序列(ssDNA)文库,通过SELEX方法,即以磁珠作为筛选介质,利用生物素-链亲和素-辣根过氧化物酶系统检测每轮ssDNA文库与CsA的亲和力,筛选针对CsA的适体,将最后一轮筛选产物克隆测序并运用相关软件进行一级结构和二级结构分析。结果:经过10轮的筛选,ssDNA文库与CsA的亲和力呈上升趋势,随机挑选的19个克隆适体根据一级结构的同源性可分为5个家族,二级结构预测以茎环(发夹)为主。结论:我们通过改良SELEX方法筛选得到了CsA特异性的适体。

Objective To screen and identify the specific aptamer for cyclosporin A（CsA） by SELEX protocol. Methods A single stranded DNA（ssDNA） random library with 78 nucleotides in length containing 35 random sequences was constructed. Biotin-streptavidin-horseradish peroxidase system was applied with magnetic beads as screening media to determine the binding affinity between ssDNA library of each round and cyclosporln A and to screen the aptamers for CsA. PCR products of the last round of screen were cloned and sequenced, and their primary and secondary structures were analyzed using related software. Result After 10 rounds of screen, an increasing tendency of binding affinity between aptamers and cyclosporin A was present. 19 randomly selected aptamer clones could be classified as 5 families based on the homology of the primary structure and the hairpin loop was the main motif in the prediction of the secondary structure. Conclusion We can obtain the specific aptamers for cyclosporin A by the establishment of SELEX technique.