毛细管电泳法筛选人IgG的Fc片段核酸适配体  

Screening of Aptamer for Human IgG Fc Fragment by Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment

作　　者：杨歌[1,2] 韩诗邈 赵丽萍 朱超 黄渊余 屈锋 YANG Ge;HAN Shi-Miao;ZHAO Li-Ping;ZHU Chao;HUANG Yuan-Yu;QU Feng(School of Life Science,Beijing Institute of Technology,Key Laboratory of Molecular Medicine and Biological Diagnosis,Ministry of Industry and Information Technology,Beijing 100081,China;Advanced Research Institute of Multidisciplinary Science,Beijing Institute of Technology,Beijing 100081,China)

机构地区：[1]北京理工大学生命学院,“分子医学与生物诊疗”工业和信息化部重点实验室,北京100081 [2]北京理工大学前沿交叉科学研究院,北京100081

出　　处：《分析化学》2020年第5期601-607,共7页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(Nos.21675012,21874010,21827810)资助。

摘　　要：基于毛细管电泳-指数富集配体系统进化技术(CE-SELEX)建立了免疫球蛋白G(IgG)的Fc片段的核酸适配体筛选方法。通过毛细管区带电泳方法分析IgG完整蛋白、Fc与Fab片段的样品的纯度、荷电性质及是否吸附的特征,并比较三者与核酸库的亲和力。结果表明,IgG与核酸库形成明显的复合物,而Fc和Fab片段与核酸库的亲和力很弱。在筛选Fc片段的适配体时,选择Fab片段作为反筛靶,对去除非特异性结合序列无显著作用。设计了针对Fc片段的适配体筛选方案,在优化的孵育条件(10 mmol/L Na2HPO4-KH 2PO 4(pH 7.17),0.05 mmol/L Mg^2+,37℃孵育25 min)下,经3轮筛选获得Fc片段的核酸适配体Seq Fc1~3,K D值为0.071~0.321μmol/L,经胶体金比色分析方法验证了其亲和力与特异性。A DNA aptamer screening method for human IgG Fc fragments was established based on capillary electrophoresis-systematic evolution of ligands by exponential enrichment(CE-SELEX).The purity,charged properties and non-adsorptivity of IgG,Fc and Fab were analyzed by capillary zone electrophoresis(CZE),and their affinity with ssDNA libraries was compared.The results showed that IgG formed significant complex with the ssDNA libraries,while Fc and Fab fragments exhibited weak affinity with ssDNA libraries.Therefore,the use of the Fab fragment as the reverse screening target had no significant effect on the sequence except for the specific binding of Fc.The aptamer screening strategy for Fc fragments was designed,the aptamers(Seq Fc1-3)were obtained under optimized incubation condition(10 mmol/L Na2HPO4-KH2PO4(pH 7.17),0.05 mmol/L Mg^2+,incubated at 37℃for 25 min)by three round of CE-SELEX,KD was 0.071-0.321μmol/L,and their affinity and specificity were verified by AuNPs colorimetry.

关 键 词：免疫球蛋白 免疫球蛋白G Fc片段 核酸适配体筛选 毛细管电泳 毛细管电泳-指数富集配体系统进化 

分 类 号：O658.9[理学—分析化学] R446.6[理学—化学]