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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.
In vitro, we synthesized a randomized single-stranded oligonucleotide (ssDNA) library with 78 nucleotides in length and 35 random sequences in the middle. Using exponential enrichment ligand system evolution (SELEX) A (CsA) was used as the target and magnetic beads were used as the screening medium. The biotin-streptavidin-HRP system was used to detect the affinity of each ssDNA library with CsA. The CsA-specific Aptamer.After 11 rounds of screening, the affinity between ssDNA library and CsA showed an upward trend.Firstly, the cloned and sequenced products from the tenth round of screening were used to analyze the primary structure and the secondary structure.The 19 randomly selected cloned aptamers, According to the primary structure of homology can be divided into five families, the secondary structure is predicted to stem ring (hairpin), which may be aptamer and CsA role of parts.CsA specific aptamer will be used for enzyme Linked method, immunofluorescence and other CsA detection.