基于毛细管电泳法-指数富集配体系统进化筛选维吉霉素M<sub>1</sub>适配体及其检测应用

引用本文: 蔡先洪, 孙强, 吴谦, 叶浩楠, 巫沁芳, 区文卉, 陈锦灿, 陈兰美. 基于毛细管电泳法-指数富集配体系统进化筛选维吉霉素M₁适配体及其检测应用[J]. 分析化学, 2022, 50(5): 728-738. doi: 10.19756/j.issn.0253-3820.210827 shu

Citation: CAI Xian-Hong, SUN Qiang, WU Qian, YE Hao-Nan, WU Qin-Fang, OU Wen-Hui, CHEN Jin-Can, CHEN Lan-Mei. Screening of Virginiamycin M₁ Aptamer Based on Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Application[J]. Chinese Journal of Analytical Chemistry, 2022, 50(5): 728-738. doi: 10.19756/j.issn.0253-3820.210827 shu

基于毛细管电泳法-指数富集配体系统进化筛选维吉霉素M₁适配体及其检测应用

蔡先洪 ^1, ,
孙强 ^1, ,
吴谦 ^1, ,
叶浩楠 ^1, ,
巫沁芳 ^1, ,
区文卉 ^1, ,
陈锦灿 ^{2,3,4,
,} ,
陈兰美 ^{1,
,}

1.
广东医科大学药学院, 湛江 524023;
2.
广东医科大学海洋医药研究院, 湛江 524023;
3.
广东湛江海洋医药研究院, 湛江 524023;
4.
南方海洋科学与工程广东省实验室, 湛江 524023

通讯作者: 陈锦灿,E-mail:jincanchen@126.com; 陈兰美,E-mail:lanmeichen@126.com

基金项目:
南方海洋科学与工程广东省实验室（湛江）重大项目（No.ZJW-2019-007）、湛江市竞争性分配项目（No.2021A05037）和2021年国家级大学生创新训练项目（No.202110571028）资助。

摘要: 毛细管电泳（Capillary electrophoresis，CE）是一种高效筛选核酸适配体的方法，具有分离效率高、样品用量少、筛选成本低等特点。本研究基于毛细管电泳-指数富集配体系统进化技术（CE-SELEX）建立了维吉霉素M₁（VGM-M₁）适配体的筛选方法。将初始ssDNA库与VGM-M₁混合孵育，通过毛细管电泳-紫外检测器（CE-UV）分离并收集结合物，通过PCR扩增和ssDNA制备，得到下一轮的子库ssDNA。经过4轮筛选后，获得富集程度较高的前10条候选适配体序列，选择富集程度最高的前4条候选序列进行亲和力及特异性评估，得到亲和力最大以及特异性最强的VGM-M₁适配体Seq 1，其平衡常数（K_d）为49 nmol/L。将适配体Seq 1进行结构修饰改造，得到剪短候选适配体序列，并对其进行亲和力以及特异性评估。结果表明，结构修饰后的适配体Seq 1-1具有更高的亲和力及特异性，其K_d低至45 nmol/L。使用竞争性酶联适配体可视化检测方法对VGM-M₁进行检测，检出限低至51 ng/mL，低于国标对VGM-M₁的最高残留限量（100 μg/L）。进一步对牛奶中VGM-M₁进行检测，加标回收率达到97.8%~103.2%。研究结果表明，基于CE-SELEX筛选的适配体Seq 1-1可特异性识别VGM-M₁，可以应用于实际样品中VGM-M₁的检测。

关键词:

English

Screening of Virginiamycin M₁ Aptamer Based on Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Application

1.
School of Pharmacy, Guangdong Medical University, Zhanjiang 524023, China;
2.
The Marine Biomedical Research Institute, Guangdong Medical University, Zhanjiang 524023, China;
3.
The Marine Biomedical Research Institute of Guangdong Zhanjiang, Zhanjiang 524023, China;
4.
Southern Marine Science and Engineering Guangdong Laboratory, Zhanjiang 524023, China

Corresponding author: CHEN Jin-Can, ; CHEN Lan-Mei,

Received Date: 04 November 2021
Revised Date: 12 February 2022
Fund Project:
Supported by the Fund of Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang) (No.ZJW-2019-007), the Science and Technology Program of Zhanjiang (No.2021A05037) and the University Student Innovation Experiment Program (No.202110571028).

Abstract: Capillary electrophoresis (CE) has been considered as an efficient method for aptamer screening due to its high separation efficiency, less sample consumption and low screening cost. In this study, an aptamer screening method was established for virginiamycin M₁ (VGM-M₁) based on capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). The initial ssDNA library was mixed and incubated with VGM-M₁, and the conjugate was separated and collected by capillary electrophoresis-ultraviolet detection (CE-UV). The next sub-library ssDNA was obtained by polymerase chain reaction (PCR) amplification and single stranded DNA (ssDNA) preparation. After four rounds of screening, the first 10 candidate aptamer sequences with high enrichment were obtained, and the first 4 candidate aptamer sequences with the highest enrichment were selected for affinity and specificity evaluation. The VGM-M₁ aptamer Seq 1 with the highest affinity and the strongest specificity was obtained, and its equilibrium constant value (K_d) was 49 nmol/L. The aptamer Seq 1 was modified to obtain optimized candidate aptamer sequences and their affinity and specificity were evaluated. The results showed that the modified aptamer Seq 1-1 had the highest affinity and specificity, and its K_d was as low as 45 nmol/L. Competitive enzyme-linked aptamer visual detection method was used to detect VGM-M₁, and the minimum detection limit was as low as 51 ng/mL, which was far below the maximum residue limit of 100 ng/mL for VGM-M₁. Further standard addition and recovery experiments were carried out on VGM-M₁ in milk, and the recoveries reached 97.8%.8-103.2%. The above research results showed that the aptamer Seq 1-1 screened by CE-SELEX could specifically recognize VGM-M₁ and could be applied to the detection of VGM-M₁ in real samples.

Key words:

Virginiamycin M₁
/ Aptamer
/ Capillary electrophoresis
/ Capillary electrophoresis-systematic evolution of ligands by exponential enrichment

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1.
沈阳化工大学材料科学与工程学院沈阳 110142

基于毛细管电泳法-指数富集配体系统进化筛选维吉霉素M₁适配体及其检测应用

通讯作者: 陈锦灿,E-mail:jincanchen@126.com; 陈兰美,E-mail:lanmeichen@126.com

English

Screening of Virginiamycin M₁ Aptamer Based on Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Application

Corresponding author: CHEN Jin-Can, ; CHEN Lan-Mei,

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

基于毛细管电泳法-指数富集配体系统进化筛选维吉霉素M1适配体及其检测应用

通讯作者: 陈锦灿,E-mail:jincanchen@126.com; 陈兰美,E-mail:lanmeichen@126.com

English

Screening of Virginiamycin M1 Aptamer Based on Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Application

Corresponding author: CHEN Jin-Can, ; CHEN Lan-Mei,

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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基于毛细管电泳法-指数富集配体系统进化筛选维吉霉素M₁适配体及其检测应用

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