SELEX技术筛选钙通道特异性核酸适配体及其抗-第三军医大学学报.doc

SELEX技术筛选钙通道特异性核酸适配体及其抗肥大细胞活化的作用 杨永强1 ，陈晓红2 ，李怡然2 ，陈 波2 ，冉新泽3 ，杨 涛1 ，孙仁山1 （400042 重庆，第三军医大学大坪医院野战外科研究所皮肤科1 ；400038重庆，第三军医大学：药学院药理学教研室2 ，军事预防医学院全军复合伤研究所，创伤、烧伤与复合伤国家重点实验室3） [摘要] 目的 筛选针对Orai1分子第1膜外区蛋白的核酸适配体，探讨适配体通过拮抗钙离子释放激活通道（Ca2+ release activation channel，CRAC）随机文库PCR对单链DNA文库进行扩增。采用聚苯乙烯酶联板为介质的指数富集配体系统进化(systematic evolution of ligand by exponential enrichment，SELEX)技术筛选核酸适配体。利用酶联免疫吸附实验检测各轮筛选序列与靶蛋白的亲和力，验证适配体与靶蛋白的特异性结合；激光共聚焦显微镜观察适配体对细胞内钙离子浓度变化的影响；利用IgE介导的肥大细胞脱颗粒模型，观察适配体对β-氨基己糖苷酶释放的抑制效应。结果 经过12轮SELEX筛选，获得了7条核酸适配体，其中AptamerY1解离常数（Kd值）为1.72×10-8mol/L。以终浓度2μg/mL的AptamerY1可抑制由IgE介导的人肥大细胞系LAD2钙离子内流，并对LAD2细胞的β-氨基己糖苷酶释放抑制率达95%。结论 通过SELEX筛选，获得了7条针对Orai1分子第1膜外区的核酸适配体，所获得适配体可对肥大细胞LAD2钙内流有效抑制，进而抑制肥大细胞活化和β-氨基己糖苷酶的释放。 [关键词] 肥大细胞；SELEX适体技术；钙离子释放激活通道；β-氨基己糖苷酶 [中图法分类号] [文献标志码] A Calcium channel-specific oligonucleotide aptamers selected by SELEX inhibit mast cell activation Yang Yongqiang1, Chen Xiaohong2, Li Yiran2, Chen Bo2, Ran Xinze3, Yang Tao1, Sun Renshan1 (1Department of Dermatology, Institute of Battle Surgery, Daping Hospital, the Third Military Medical University, Chongqing 400042, China; 2Department of Pharmacology, College of Pharmacy, 3Institute of Combined Injury, College of Military Preventive Medicine, the Third Military Medical University, Chongqing, 400038, China) [Abstract] Objective The oligonucleotide aptamers for the first extracellular domain of Orai1 protein were obtained and characterized for exploring the inhibition effects of aptamers on mast cells activation by Ca2+ release activation channel (CRAC). Methods Random single-strand DNA (ssDNA) library was constructed. The key parameters of asymmetric PCR were optimized to amplify ssDNA library. The oligonucleotide aptamers were screened by systematic evolution of ligand by exponential enrichment (SELEX) technology with enzyme-linked polystyrene plate as medium. The affinity between ssDNA library and target protein was examined in each round by ELIS