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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (12): 41-48    DOI: 10.13523/j.cb.20181206
Orginal Article     
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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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     
Received: 27 June 2018      Published: 10 January 2019
ZTFLH:  Q78  
Corresponding Authors: Jia WANG     E-mail: wangjia@mail.hzau.edu.cn
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Qian PANG
Jing CHEN
Xiao-hong WANG
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Cite this article:

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.

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

Fig.1 Amplifying VH and VL gene and construction of scFv
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
Table 1 The enrichment of phage display anti-AFB1 scFv by immunoaffinity panning
Fig.2 Amino acid sequences of anti-AFB1 scFv (underlined part was the Linker sequence)
Fig.3 SDS-PAGE analysis for expression of scFv-A/B M:Protein marker; 1:Expression of scFv
Fig.4 3D model of anti-AFB1 scFv (a) and its molecular docking model with AFB1 (b)
Fig.5 2D molecular docking model scFv and AFB1
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
Table 2 Comparison of the binding mode of different antibodies to AFB1
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