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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (12): 41-48    DOI: 10.13523/j.cb.20181206
技术与方法     
基于噬菌体展示技术抗黄曲霉毒素B1单链抗体的筛选及其蛋白结构分析 *
庞倩,陈晶,王小红,王佳()
华中农业大学食品科技学院 武汉 430070
Screening of Anti-Aflatoxin B1 ScFv Based on Phage Display Technology and Analysis of Its Protein Structure
PANG Qian,CHEN Jing,WANG Xiao-hong,WANG Jia()
College of Food Science and technology, Huazhong Agricultural University, Wuhan 430070, China
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摘要：

黄曲霉毒素B1(aflatoxin B1,AFB1)是一种毒性强、污染广的真菌毒素,建立高效、准确、快速的AFB1检测方法具有重要意义。利用噬菌粒-辅助噬菌体展示系统构建单链抗体(single chain variable fragment,scFv)文库,应用“淘选-洗脱”的策略,是筛选高亲和配体的常用方法之一。同时结合同源建模和分子对接等计算机辅助手段,分析得到抗体与抗原的结合位点与关键氨基酸,为在基因水平改造抗体提供基础。从AFB1-BSA免疫小鼠的脾细胞内扩增重链可变区和轻链可变区,将组合成的scFv片段插入噬菌粒pCANTAB5e中,构建了噬菌体展示单链抗体文库,以不同浓度的AFB1-OVA作为抗原,从文库中筛选到一株亲和力较好的抗AFB1单链抗体scFv,其亲和常数为8×10 5L/mol;根据同源建模和分子对接发现,与抗原AFB1结合时,scFv中Tyr33、Ser52和Tyr102起关键作用,分别以π-π共轭键、氢键和范德华力与AFB1结合。

关键词： 黄曲霉毒素B1;  单链抗体;  噬菌粒-辅助噬菌体展示;  同源建模;  分子对接    
Abstract:

Aflatoxin B1 (AFB1) is a mycotoxin which has highly toxic and highly polluted. It is of great significance for AFB1 to establish an efficient, accurate and rapid detection method. Phagemid/helper phage system is one of the most common used system for single-chain antibody fragment(scFv) libraries construction. The “panning-elution” strategy is an effective method for screening ligands with high affinity. Combined with homology modeling and molecular docking, the key amino acid binding sites between antibody and antigen were analyzed to provide the basis for genetically modifying antibodies. In this study, the heavy chain variable regions and light chain variable regions were amplified from the spleen cells of AFB1-BSA immunized mice. Then assembled scFv fragment was inserted into the phagemid pCANTAB5e to construct a phage display single-chain antibody library. Using different concentrations of AFB1-OVA as coating antigens, anti-AFB1 scFv was isolated from this library, which affinity constant is 8×10 5L/mol. According to homology modeling and molecular docking, Tyr33, Ser52, and Tyr102 play a key role in binding with AFB1 under π-π conjugated bonds, hydrogen bonds, and van der Waals forces, respectively.

Key words: AFB1    Single-chain antibody fragment    Phagemid/helper phage display    Homology modeling    Molecular docking
收稿日期: 2018-06-27 出版日期: 2019-01-10
ZTFLH:  Q78  
基金资助: * 国家自然科学基金(31601539);* 湖北省自然科学基金(2016CFB143)
通讯作者: 王佳     E-mail: wangjia@mail.hzau.edu.cn
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引用本文:

庞倩,陈晶,王小红,王佳. 基于噬菌体展示技术抗黄曲霉毒素B1单链抗体的筛选及其蛋白结构分析 *[J]. 中国生物工程杂志, 2018, 38(12): 41-48.

PANG Qian,CHEN Jing,WANG Xiao-hong,WANG Jia. Screening of Anti-Aflatoxin B1 ScFv Based on Phage Display Technology and Analysis of Its Protein Structure. China Biotechnology, 2018, 38(12): 41-48.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181206    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I12/41

图1  可变区VH与VL基因扩增与scFv构建
筛选次数 AFB1-OVA浓度(μg/ml) 噬菌体投入量(cfu/ml) 噬菌体捕获量(cfu/ml) 富集度
1 20 2.1×1013 5×102 -
2 20 1.3×1013 9×102 2.875
3 20 1.5×1013 1.2×103 1.153
4 10 2.5×1013 1.6×103 0.8
5 10 1.8×1013 3×103 2.61
表1  免疫亲和筛选对抗AFB1噬菌体单链抗体的富集
图2  抗AFB1 scFv的氨基酸序列(划线处为Linker)
图3  scFv-A/B纯化后的SDS-PAGE分析
图4  scFv-AFB1三维模型图(a)及与AFB1的分子对接结构图(b)
图5  scFv-AFB1分子对接2D图
抗体名称 抗体类型 构建方法 与AFB1的结合方式 参考文献
Nb-G8 纳米抗体 噬菌体展示纳米抗体免疫文库 Thr32、Ile33、Phe49、Typ54 、Tyr106 和 Val112 等氨基酸残基可能直接参与抗原识别表位的形成 [32]
scFv-H4 单链抗体 Tomlinson I+J文库 AFB1主要被轻链上的Ala91、Pro95、Phe98和重链上的Lys96、Thr97、His95构成疏水腔包裹,双呋喃环与Asp之间形成氢键 [33]
1C11 单克隆抗体 杂交瘤细胞株 1C11重链上的Ser49和Phe103以氢键与疏水作用与AFB1结合 [34]
scFv 单链抗体 抗AFB1单克隆抗体杂交瘤细胞株 在三级结构中连接肽将VH和VL区域牵拉而相互靠近,形成典型沟槽结构,是scFv的抗原结合区域 [35]
scFv 单链抗体 噬菌粒-辅助噬菌体展示系统构建的单链抗体文库 与抗原AFB1结合时,scFv中Tyr33、Ser52和Tyr102起关键作用,分别以π-π共轭键、氢键和范德华力与AFB1结合 本研究
表2  不同类型的抗体与AFB1结合方式的比较
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