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引用本文:   杨歌, 赵毅, 韩诗邈, 朱超, 黄渊余, 屈锋. 毛细管电泳法筛选脱铁转铁蛋白的核酸适配体及筛选影响因素分析. 分析化学, 2020, 48(5): 632-641. doi:  10.19756/j.issn.0253-3820.191694 [复制]

Citation:   YANG Ge , ZHAO Yi , HAN Shi-Miao , ZHU Chao , HUANG Yuan-Yu , QU Feng . Screening Aptamer of Apo-transferrin via Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Environmental Factors Analysis. Chinese Journal of Analytical Chemistry, 2020, 48(5): 632-641. doi: 10.19756/j.issn.0253-3820.191694 [复制]

毛细管电泳法筛选脱铁转铁蛋白的核酸适配体及筛选影响因素分析

收稿日期: 2019-11-25

基金项目: 本文系国家自然科学基金项目(Nos.21675012,21874010,21827810)资助

Screening Aptamer of Apo-transferrin via Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Environmental Factors Analysis

Received Date:  2019-11-25

Fund Project:  This work was supported by the National Natural Science Foundation of China (Nos. 21675012, 21874010, 21827810).

核酸适配体是通过指数富集配体系统进化(SELEX)技术,在体外筛选获得的与目标分子有高亲和力与特异性的寡核苷酸序列。由于筛选过程周期长且影响因素复杂,使用常规筛选方法进行多因素影响的条件优化的工作量大,样品消耗多,目前还少有系统的研究报道。毛细管电泳(CE)具有高分辨率、快速分离、样品用量小、筛选成本低的优势,是核酸适配体筛选的有效方法。本研究以人血清中脱铁转铁蛋白(A-TF)为模式蛋白,利用CE方法研究核酸库长度、孵育温度、缓冲溶液的种类与pH值、金属离子等因素对靶蛋白与核酸库相互作用的影响。结果表明,较短序列的核酸库与靶蛋白的亲和力更高;孵育温度、缓冲液种类与pH值均影响靶蛋白与核酸复合物的形成;低浓度的K、Ca2+与Mg2+可促进复合物形成。基于优化的筛选条件,经过3轮毛细管电泳法筛选,获得了A-TF的适配体Seq A3,采用CE-LIF测得复合物的亲和常数KD=0.476 μmol/L。此适配体可用于人血清基质中A-TF的识别。

关键词:   脱铁转铁蛋白, 核酸适配体筛选, 影响因素, 毛细管电泳, 指数富集配体系统进化
Key words:   Apo-transferrin, Aptamer screening, Influencing factors, Capillary electrophoresis, Systematic evolution of ligands by exponential enrichment
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毛细管电泳法筛选脱铁转铁蛋白的核酸适配体及筛选影响因素分析

杨歌, 赵毅, 韩诗邈, 朱超, 黄渊余, 屈锋

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