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设计并合成随机ssDNA文库,以硝酸纤维素膜为筛选介质,利用SELEX技术筛选人Cε3-Cε4蛋白特异性高亲和力核酸适配子,通过对退火温度、循环次数及靶蛋白与ssDNA摩尔比等关键参数进行优化,建立了适合的筛选体系。ELISA测定各适配子亲和力,获得了人Cε3-Cε4蛋白高亲和力、高特异性适配子,并分析了其一级结构和二级结构。以所获高亲和力适配子分别作为捕获适配子和检测适配子,建立了酶联适配子吸附试验(enzyme-linked aptamers sorption assay,ELASA)方法,灵敏度达到120 ng.mL-1,可用于人IgE的定量检测。
The random ssDNA library was designed and synthesized. The nitrocellulose membrane was used as the screening medium. SELEX technology was used to screen human Cε3-Cε4 protein-specific high-affinity aptamers. Through annealing temperature, number of cycles and molar ratio of target protein to ssDNA Parameter optimization, established a suitable screening system. The affinity of each aptamer was measured by ELISA, and the high affinity and high specificity aptamers of human Cε3-Cε4 protein were obtained. The primary and secondary structures of the human Cε3-Cε4 protein were also analyzed. The high-affinity aptamers were used as capture aptamers and aptamer aptamers, respectively. The enzyme-linked aptamers sorption assay (ELASA) was used to establish a sensitivity of 120 ng.mL-1, Can be used for quantitative detection of human IgE.