CA125抗原核酸适体的筛选及鉴定  

Screening and identification of aptamers to CA125 antigen

作　　者：黄百海 何雷 李保林 修尘林 赵运旺 HUANG Baihai;HE Lei;LI Baolin;XIU Chenlin;ZHAO Yunwang(College of Environment and Chemical Engineering,Yanshan University,Qinhuangdao 066004,China;The First Hospital of Qinhuangdao,Qinhuangdao 066000,China)

机构地区：[1]燕山大学环境与化学工程学院,河北秦皇岛066004 [2]秦皇岛市第一医院,河北秦皇岛066000

出　　处：《分子影像学杂志》2019年第1期52-56,共5页Journal of Molecular Imaging

基　　金：国家自然科学基金(30960104;30770639)

摘　　要：目的建立一种基于核酸适体快速检测肿瘤血清标志物CA125抗原的方法。方法以CA125抗原为筛选靶标,CA199抗原为反筛选靶标,羧基化琼脂磁珠为筛选介质,利用指数级富集配体的系统进化技术筛选其高特异性核酸适体,利用生物信息学方法对核酸适体进行序列分析和二级结构预测,荧光定量法分析核酸适体的亲和力,ELONA法测定核酸适体的特异性。结果经过6轮消减SELEX筛选,次级ssDNA文库的亲和力逐渐提高,特异性次级ssDNA文库逐轮富集,经二代测序,获得3条拷贝数>1000的核酸序列(Apt-1、Apt-2、Apt-3),可特异性识别CA125抗原,测定Kd值,分别为41.02±4.9、19.06±9.0、10.02±5.0 nmol/L,Apt-3亲和力最高。结论功能性核酸适体Apt-3能高特异性识别CA125抗原,为肿瘤血清标志物CA125检测提供了一种新的、快速、有效的工具和手段。Objective To establish a method for rapid detection of tumor serum marker CA125 antigen based on aptamer.Methods CA125 antigen was used as the screening target,CA199 antigen was used as the anti-screening target,and carboxylated agar magnetic beads were used as the screening medium.The phylogenetic technique of exponential enrichment ligand was used to screen the high specificity aptamer,and the bioinformatics method was used.The sequence analysis and secondary structure prediction of the aptamer were carried out,the affinity of the aptamer was analyzed by fluorescence quantification,and the specificity of the aptamer was determined by the ELONA method.Results After 6 rounds of SELEX screening,the affinity of the secondary ssDNA library was gradually increased,and the specific secondary ssDNA library was enriched round-by-round.After sequencing by the second generation,three nucleic acid sequences with copy number>1000 were obtained(Apt-1,Apt-2,Apt-3),can specifically recognize CA125 antigen,and determine Kd values,which are 41.02±4.9 nmol·L^–1,19.06±9.0 nmol·L^–1,10.02±5.0 nmol·L^–1,Apt-3 the highest affinity.Conclusion The functional aptamer Apt-3 can highly recognize CA125 antigen,which provides a new,rapid and effective tool and means for the detection of tumor serum marker CA125.

关 键 词：CA125抗原 消减SELEX技术 核酸适体 高通量测序技术 

分 类 号：R730.43[医药卫生—肿瘤]