筛选环孢霉素A适体的SELEX技术的建立(英文)

In vitro Selection of DNA Aptamer to Cyclosporin A by SELEX Method

体外合成一个全长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,运用指数富集的配体系统进化(SELEX)技术,以环孢霉素A(CsA)为靶目标,以磁珠作为筛选介质,利用生物素-链酶抗生物素-辣根过氧化物酶系统,检测每轮ssDNA文库与CsA的亲和力,筛选并鉴定CsA特异性的适体.经过11轮的筛选,ssDNA文库与CsA的亲和力呈上升趋势.将第10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.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,这可能是适体与CsA作用的部位.CsA特异性的适体将用于酶联法、免疫荧光法等对CsA进行检测.

To obtain the aptamers with high affinity and specificity for cyclosporin A （CsA）, a synthesized 78 nt single stranded DNA （ssDNA） random library containing 35 random sequences flanked by invariant primer was subjected to 11 rounds of selection against CsA by SELEX protocol. Magnetic beads were used for target immobilization and the biotin-streptavidin-horseradish peroxidase system was employed for determining the binding affinity between the aptamers and CsA. After ten rounds of selection and amplification, with an increasing affinity for each round, the selected aptamers were cloned, sequenced and analyzed for their primary and secondary structures. The 19 aptamers were divided into five groups based on primary sequence homology. Hairpin loop is the main motif in the predicted secondary structure and is supposed to be the binding part of the aptamers to CsA. The CsA-specific aptamers will be useful for enzyme-linked assays or immunofluorescence assays of CsA.