人FcγRI特异性核酸适配体的体外筛选及鉴定

In vitro Selection and Identification of Aptamers Against Human FcγRI

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 FcγRI （i. e. , CD64） , one of the high affinity Fc receptors for IgG, is a new specific and sensitive biomarker for early diagnosis of sepsis and systematic bacterial infection. Expression of FcγRI is presently determined by flow cytometry analysis, which is not available for most laboratories. This study aims to select aptamers against human FcγRI with high affinity and specificity from a synthesized random oligonucleotide library by a novel in vitro selection technique, termed systematic evolution of ligands by exponential enrichment （SELEX） protocol. Human FcγRI immobilized on carboxytic acid magnetic beads was used as the target for eight rounds of selection of aptamers by SELEX, and three key monoclonal aptamers against FcγRI were obtained. Bioinformatics analysis showed that stem-loop and G-quartet are the main predicted secondary structures of aptamers to human FcγRI, suggesting the potential binding sites between FcγRI and aptamers, and G-T mismatch is also common. Flow cytometry and fluorescence microscopy assays respectively demonstrated that equilibrium dissociation constant （Kd） for the selected key monoclonal aptamers are in the nanomolar range, and aptamers only recognize neutrophils from the patient with sepsis, indicating their good affinity and specificity. Our results suggest that specific aptamers against humanFcγRI have been successfully screened by SELEX. This study provides valuable information for further development of new detection methods based on aptamer molecular probes for early laboratory diagnosis of sepsis.