人FcγRI特异性核酸适配体的体外筛选及鉴定

发布时间：2018-03-31 03:18

本文选题：指数富集配基的系统进化技术　切入点：核酸适配体　出处：《中国生物化学与分子生物学报》2017年08期

【摘要】：人中性粒细胞FcγRI(即CD64),Ig G Fc高亲和力受体之一,是早期诊断脓毒血症和系统性细菌感染的一个灵敏和特异的新标志物;目前,采用流式细胞计量术测定,难以在一般实验室开展。本研究旨在应用新型的体外筛选技术——指数富集配基的系统进化(systematic evolution of ligands by exponential enrichment,SELEX)技术——从体外合成的随机寡核苷酸文库中筛选人FcγRI的高亲和力和高特异性的核酸适配体(aptamer)。本文以人FcγRI为靶标,将其固定在羧基活化磁珠上,进行SELEX筛选。经过8轮筛选,共获得3个重点研究的单克隆适配体。生物信息学分析结果显示,人FcγRI适配体的模拟二级结构以茎-环和G-四聚体为主,可能是FcγRI与适配体的作用位点,G-T错配常见。流式细胞计量术和荧光显微镜分析显示,筛选出的典型单克隆适配体解离常数(the dissociation constant,Kd)均达到纳摩尔水平,而且适配体只与脓毒血症中性粒细胞结合,具有良好的亲和力和特异性。本研究表明,通过SELEX技术,成功获取了人FcγRI特异性核酸适配体,为以此适配体为分子探针,进一步建立用于脓毒血症早期诊断的新方法奠定了基础。
[Abstract]:Human neutrophil FC 纬 RI, one of the high affinity receptors of Ig G FC, is a sensitive and specific new marker for early diagnosis of sepsis and systemic bacterial infection. The aim of this study was to apply a new in vitro screening technique, the systematic evolution of ligands by exponential oligonucleotide Selex, to screen the high level of human FC 纬 RI from a random oligonucleotide library synthesized in vitro. Affinity and high specificity of aptamer.Human FC 纬 RI was used as the target. It was immobilized on carboxyl activated magnetic beads for SELEX screening. After 8 rounds of screening, three major monoclonal aptamers were obtained. Bioinformatics analysis showed that, The simulated secondary structures of human FC 纬 RI aptamers are mainly stem-ring and G-tetramer, which may be the common G-T mismatch between FC 纬 RI and aptamer. The typical monoclonal aptamer dissociation constant of the dissociation constantKd) has reached the nanomolor level, and the aptamer only binds to septic neutrophils with good affinity and specificity. The specific aptamer of human FC 纬 RI nucleic acid was successfully obtained, which laid a foundation for the further establishment of a new method for the early diagnosis of sepsis by using the aptamer as a molecular probe.
【作者单位】：福州总医院检验科;
【基金】：福建省科技计划项目(No.2012D026) 福建省社会发展重点项目(No.2010Y0044)资助~~
【分类号】：R446.6;R459.7

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