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抗噻虫嗪重组全长抗体的制备与特异性识别机制研究

刘鹏琰 郭源昊 焦沙沙 陈阳 郭逸蓉 朱国念

刘鹏琰, 郭源昊, 焦沙沙, 陈阳, 郭逸蓉, 朱国念. 抗噻虫嗪重组全长抗体的制备与特异性识别机制研究[J]. 农药学学报, 2021, 23(2): 296-307. doi: 10.16801/j.issn.1008-7303.2021.0008
引用本文: 刘鹏琰, 郭源昊, 焦沙沙, 陈阳, 郭逸蓉, 朱国念. 抗噻虫嗪重组全长抗体的制备与特异性识别机制研究[J]. 农药学学报, 2021, 23(2): 296-307. doi: 10.16801/j.issn.1008-7303.2021.0008
Pengyan LIU, Yuanhao GUO, Shasha JIAO, Yang CHEN, Yirong GUO, Guonian ZHU. Development of anti-thiamethoxam full-length recombinant antibody and investigation of the specific recognition mechanism[J]. Chinese Journal of Pesticide Science, 2021, 23(2): 296-307. doi: 10.16801/j.issn.1008-7303.2021.0008
Citation: Pengyan LIU, Yuanhao GUO, Shasha JIAO, Yang CHEN, Yirong GUO, Guonian ZHU. Development of anti-thiamethoxam full-length recombinant antibody and investigation of the specific recognition mechanism[J]. Chinese Journal of Pesticide Science, 2021, 23(2): 296-307. doi: 10.16801/j.issn.1008-7303.2021.0008

抗噻虫嗪重组全长抗体的制备与特异性识别机制研究

doi: 10.16801/j.issn.1008-7303.2021.0008
基金项目: 国家自然科学基金 (31871994);国家重点研发计划 (2017YFF0210200)
详细信息
    作者简介:

    刘鹏琰,女,博士研究生,主要从事农药残留免疫快速检测研究,E-maillpyainimen@163.com

    通讯作者:

    郭逸蓉,通信作者 (Author for correspondence),女,副教授,主要从事农药残留免疫快速检测研究,E-mailyirongguo@zju.edu.cn

  • 中图分类号: Q78; TP391.75; TQ450.263

Development of anti-thiamethoxam full-length recombinant antibody and investigation of the specific recognition mechanism

  • 摘要: 本研究旨在制备抗噻虫嗪的重组抗体,并采用计算机辅助的同源建模和分子对接的方法解析抗体和噻虫嗪的特异性分子识别机制。首先,采用表面等离子共振技术评价了抗噻虫嗪单克隆抗体的识别性能;其次,以抗噻虫嗪杂交瘤细胞株为基因来源,经分子克隆获得了抗体可变区序列,由哺乳动物细胞HEK 293(F) 体外表达成功获得了全长重组抗体;最后,基于正确的可变区序列,采用同源建模和分子对接手段,研究了抗体高亲和力特异结合噻虫嗪的分子识别机制。结果表明,抗噻虫嗪单克隆抗体可特异性识别噻虫嗪,且与其具有较高的结合亲和力 (解离平衡常数KD = 7.995 × 10−11 mol/L)。由HEK 293(F) 体外表达的全长重组抗体,采用间接竞争酶联免疫吸附分析方法进行评价,表明该重组全长抗体对噻虫嗪的IC50值为0.41 μg/L,与其他新烟碱类农药的交叉反应率 < 0.04%,表现出与亲本单克隆抗体一致的性能,即具有高特异性、高灵敏度的识别活性。分子对接计算结果表明,位于疏水结合口袋参与形成范德华力的8个氨基酸残基和参与形成氢键的Asn39 (L-CDR1) 残基与抗体的选择性 (特异性) 相关,位于重链CDR区的两个氨基酸His35(H-CDR1) 和Trp108(H-CDR3) 残基决定了抗体对噻虫嗪的结合亲和力。该研究制备的重组全长抗体可代替传统单克隆抗体建立多种免疫检测方法,应用于环境样品和农产品中噻虫嗪的残留检测。解析的噻虫嗪抗原抗体分子识别机制,可为后续改造更高亲和力的抗体提供理论依据。
  • 图  1  采用SPR测试噻虫嗪mAb-E7E2的结合选择性

    Figure  1.  Binding selectivity of anti-thiamethoxam mAb-E7E2 determined by SPR

    图  2  SPR分析噻虫嗪mAb-E7E2对噻虫嗪和噻虫胺的亲和力和动力学

    Figure  2.  Affinity and kinetics analysis of mAb-E7E2 to thiamethoxam and clothianidin measured by SPR

    图  3  噻虫嗪mAb-E7E2的VH和VL基因以及氨基酸序列

    蓝色-CDR1 (blue-complementarity determining region 1);绿色-CDR2 (green-complementarity determining region 2);黄色-CDR3 (yellow-complementarity determining region 3).

    Figure  3.  Gene and amino acid sequences of VH and VL of anti-thiamethoxam mAb-E7E2

    图  4  (A) 含有表达载体同源臂的重链和κ轻链可变区片段核酸凝胶电泳,M是5000 DNA marker;(B) 哺乳动物细胞表达全长重组抗体的SDS-聚丙烯酰胺凝胶电泳图

    Figure  4.  (A) VH/Vκ fragments with homologous arms of expression plasmid. M: 5000 DNA Marker map; (B) SDS-PAGE of full-length rAb expressed in HEK 293 cells

    图  5  基于IC-ELISA法建立单克隆抗体和全长重组抗体检测噻虫嗪的标准曲线 (n=3)

    Figure  5.  Standard curves of thiamethoxam established by ic-ELISAs based on mAb-E7E2 and full-length rAb (n=3)

    图  6  (A) 噻虫嗪mAb-E7E2可变区空间结构模型a;(B) 模型质量评价的Ramachandran图;(C) 模型质量评价的Verify3D图

    抗体可变区以条带显示,颜色分别为深绿色 (FR)、紫色 (CDR-L1/L2/L3)、棕色 (CDR-H1/H2) 和红色 (CDR-H3)。

    Figure  6.  (A) 3D structure model of variable region of thiamethoxam mAb-E7E2; (B) Ramachandran plot for model quality validation; (C) Verify3D score plot for model quality validation

    Variable regions of antibody were shown in ribbons and colored in dark green (Framework, FR), purple (VL-CDR1/2/3), brown (VH-CDR1/2) and red (VH-CDR3).

    图  7  抗体可变区模型与噻虫嗪或噻虫胺对接复合物的二维结合模式图和三维空间结构图

    (A1) 抗体可变区与噻虫嗪形成稳定复合物存在的主要作用力和关键氨基酸的二维平面图;(A2) 抗体可变区与噻虫嗪预测结合模式的三维空间图;(B1) 抗体可变区与噻虫胺形成稳定复合物存在的主要作用力和关键氨基酸的二维平面图;(B2) 抗体可变区与噻虫胺预测结合模式的三维空间图。

    Figure  7.  The 2D binding mode and 3D structure of the docked complex of modeled variable region

    (A1) The 2D diagram of the key amino acid residues and main interactions that contributed to the stability of antibody-thiamethoxam complex. (A2) The 3D diagram of the predicted binding mode of thiacloprid to the variable fragment (Fv). (B1) The 2D diagram of the key amino acid residues and main interactions that contributed to the stability of antibody-clothianidin complex. (B2) The 3D diagram of the predicted binding mode of clothianidin to the variable fragment (Fv).

    表  1  亚型特异性引物用于扩增重/轻链可变区基因序列

    Table  1.   Subtype-specific primers used to amplify the VH andVL gene sequences

    基因名称
    Gene name
    上游引物
    Forward primer 5′-3′
    下游引物
    Reverse primer 5′-3′
    VH 1: ATGAACTTYGGGYTSAGMTTGRTTT
    2: ATGTACTTGGGACTGAGCTGTGTAT
    3: ATGAGAGTGCTGATTCTTTTGTG
    4: ATGGATTTTGGGCTGATTTTTTTTATTG
    5: CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG
    1.CCAGGGRCCARKGGATARACIGRTGG
    2.ACTAGTCGACATGGRATGGASCKKIRTCTTTMTCT
    VL 1: ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT
    2: ACTAGTCGACATGGATTTWCARGTGCAGATTWTCAGCTT
    3: ACTAGTCGACATGGTYCTYATVTCCTTGCTGTTCTGG
    4: ACTAGTCGACATGGTYCTYATVTTRCTGCTGCTATGG
    5: CCCAAGCTTACTGGATGGTGGGAAGATGGA
    1.ACTGGATGGTGGGAAGATGGA
    2.ACTAGTCGACATGGATTTWCARGTGCAGATTWTCAGCTT
    下载: 导出CSV

    表  2  噻虫嗪全长重组抗体与亲本抗体交叉反应率的比较

    Table  2.   Cross-reactivity (CR) of thiamethoxam and analogues determined by ic-ELISA based on full-length rAb and mAb-E7E2

    化合物  
    Compound  
    分子结构
    Structural formula
    交叉反应率 CR/%
    亲本单
    克隆抗体
    Parental
    mAb
    全长重组
    抗体
    Full-length
    rAb
    噻虫嗪 thiamethoxam 100 100
    噻虫胺 clothianidin 0.04 0.04
    N-去甲基噻虫嗪
    N-demethyl-thiamethoxam
    0.02 0.03
    氯噻啉 imidaclothiz 0 0
    吡虫啉 imidacloprid 0 0
    噻虫啉 thiacloprid 0 0
    啶虫脒 acetamiprid 0 0
    烯啶虫胺 nitenpyram 0 0
    呋虫胺 dinotefuran 0 0
    下载: 导出CSV

    表  3  抗噻虫嗪抗体与噻虫嗪和噻虫胺的分子对接计算结果

    Table  3.   Computer aided molecular docking results of anti-thiamethoxam antibody with thiamethoxam and clothianidin

    农药
    Pesticide
    结合自由能打分
    Binding energy score/(kJ/mol)
    主要作用力
    Main interactions
    氨基酸
    Amino acids
    区域
    Regions
    噻虫嗪 thiamethoxam −22.91 H-bond (0.338 nm) Asn39 L-CDR1
    CH-Pi (0.345 nm) His35 H-CDR1
    CH-Pi (0.457 nm) Trp108 H-CDR3
    VDW Trp33 H-CDR1
    VDW Gly99 H-CDR3
    VDW Trp108 H-CDR3
    VDW Tyr37 L-CDR1
    VDW Val94 L-CDR3
    VDW Gln95 L-CDR3
    VDW Gly96 L-CDR3
    VDW His101 L-CDR3
    噻虫胺 clothianidin −20.86 H-bond (0.305 nm) Asn39 L-CDR1
    VDW His35 H-CDR1
    VDW Trp108 H-CDR3
    VDW Trp33 H-CDR1
    VDW Gly99 H-CDR3
    VDW Tyr37 L-CDR1
    VDW Leu55 L-CDR2
    VDW Val94 L-CDR3
    VDW Gln95 L-CDR3
    VDW Gly96 L-CDR3
    VDW His101 L-CDR3
    下载: 导出CSV

    表  4  本研究抗噻虫嗪抗体与已报道抗体性能比较

    Table  4.   Comparison of the sensitivity of anti-thiamethoxam Ab with other reported Abs

    抗体类型
    Antibodies
    检测方法     
    Detection methods     
    检出限
    Limit of
    detection/
    (μg/L)
    半抑制
    浓度
    IC50/
    (μg/L)
    交叉反
    应率
    CR/%
    年份,
    参考文献
    Year,
    references
    多克隆抗体 pAb 直接竞争酶联免疫吸附分析 dc-ELISA 0.10 8.00 < 2.0 2003,[7]
    单克隆抗体 mAb 间接竞争酶联免疫吸附分析 ic-ELISA 0.20 < 0.1 2006,[1]
    自动流式荧光免疫分析 Automated flow fluorescent immunoassay 0.016 0.03
    单克隆抗体 mAb 间接竞争酶联免疫吸附分析 ic-ELISA 0.05 0.57 < 0.1 2009,[21]
    单克隆抗体 mAb 间接竞争酶联免疫吸附分析 ic-ELISA 0.05 0.50 < 0.06 2010,[22]
    单克隆抗体 mAb 直接竞争酶联免疫吸附分析 dc-ELISA 0.10 0.72 < 1.80 2013,[23]
    单克隆抗体 mAb 间接竞争酶联免疫吸附分析 ic-ELISA 0.87 < 0.06 2018,[6]
    单克隆抗体 mAb 间接竞争酶联免疫吸附分析 ic-ELISA 0.13 0.48 < 0.04 本研究 in this study
    重组全长抗体 Full-length rAb 间接竞争酶联免疫吸附分析 ic-ELISA 0.10 0.41 < 0.04 本研究 in this study
    下载: 导出CSV
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  • 收稿日期:  2020-07-16
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