CAJ | 학술논문

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(SELEZ)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.链酶抗生物素一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.
체외합성일개전장78개핵감산,중간함35개수궤서렬적수궤단련과핵감산서렬(ssDNA)문고,운용지수부집적배체계통진화(SELEZ)기술,이배포매소A(CsA)위파목표,이자주작위사선개질,이용생물소.련매항생물소일랄근과양화물매계통,검측매륜ssDNA문고여CsA적친화력,사선병감정CsA특이성적괄체.경과11륜적사선,ssDNA문고여CsA적친화력정상승추세.장제10륜사선산물극륭측서병운용상관연건진행일급결구화이급결구분석.수궤도선적19개극륭괄체,근거일급결구적동원성가분위5개가족,이급결구예측이경배(발협)위주,저가능시괄체여CsA작용적부위.CsA특이성적괄체장용우매련법、면역형광법등대CsA진행검측.
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 1 1 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 primaryand 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 asses of CsA.