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Identifying the Epitopes of Bacillus thuringiensis Cry2Aa Toxin Involved in Cadherin Interaction by a Monoclonal Antibody

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Abstract

Antibodies are a useful tool for assistance to map the binding epitopes in Bacillus thuringiensis Cry toxins and their receptors, and even determine how receptors promote toxicity. In this work, a monoclonal antibody (mAb-1D2) was produced by the hybridoma cell line raised against Cry2Aa toxins, with a half inhibition concentration (IC50) of 9.16 μg/mL. The affinity constant of two recombinant toxin-binding fragments derived from Helicoverpa armigera and Plutella xylostella cadherin-like protein (HaCad-TBR or PxCad-TBR) to Cry2Aa toxin was measured to be 1.21 μM and 1.24 μM, respectively. Competitive ELISA showed that mAb-1D2 competed with HaCad-TBR or PxCad-TBR binding to Cry2Aa. Meanwhile, the toxicity of the Cry2Aa toxin to the H. armigera and P. xylostella larvae were greatly reduced when the toxin was mixed with mAb-1D2, which indicated that cadherin may play an important functional role in the toxicity of Cry2Aa. After transforming mAb-1D2 to a single-chain variable fragment (scFv), the hot spot residues of Cry2Aa with 1D2-scFv, PxCad-TBR, and HaCad-TBR were analyzed by molecular docking. It was demonstrated that the hot spot residues of Cry2Aa involving with 1D2-scFv interaction were mainly in Domain II, and some residues in Domain I. Moreover, mAb-1D2 and the two cadherin fragments shared the common hot spot residues on Cry2Aa, which could explain mAb-1D2 inhibited Cry2Aa binding with cadherin fragments. This monoclonal antibody could be a useful tool for identifying the binding epitopes between Cry2Aa and cadherin, and even assist to analyze the roles of cadherin in Cry2Aa toxicity.

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Funding

This work was supported by the National Natural Science Foundation of China (31972292, 31972349 and 31630061), Jiangsu Agricultural Science and Technology Innovation Fund of China (CX[20]3053).

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Authors and Affiliations

  1. College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China

    Cheng Shen, Yi-hang Li, Jiang-zhao Zhang, Jing Wang, Yuan Liu & Xian-jin Liu

  2. Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China

    Cheng Shen, Yi-hang Li, Man-man Lin, Jiang-zhao Zhang, Jing Wang, Mei-jing Gao, Xiao Zhang, Xiao-dan Hu, Yuan Liu & Xian-jin Liu

Authors
  1. Cheng Shen
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  2. Yi-hang Li
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  3. Man-man Lin
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  4. Jiang-zhao Zhang
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  5. Jing Wang
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  6. Mei-jing Gao
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  7. Xiao Zhang
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  8. Xiao-dan Hu
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  9. Yuan Liu
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  10. Xian-jin Liu
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Contributions

CS: methodology, data curation, writing original draft. YHL: formal analysis, investigation. MML: writing—review and editing. JZZ: formal analysis, investigation. JW: formal analysis, investigation. MJG: formal analysis, investigation. XZ: funding acquisition. XDH: formal analysis, investigation. YL: project administration. XJL: project administration.

Corresponding authors

Correspondence to Yuan Liu or Xian-jin Liu.

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Ethical Approval

The Medical Comparison Center of Yangzhou University (China) provided BALB/c mice for this experiment. All of the experimental protocols were approved by the animal experiment ethics committee of author’s research institute from the laboratory for food quality and safety-state key laboratory cultivation base of ministry of science and Technology.

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Shen, C., Li, Yh., Lin, Mm. et al. Identifying the Epitopes of Bacillus thuringiensis Cry2Aa Toxin Involved in Cadherin Interaction by a Monoclonal Antibody. Appl Biochem Biotechnol 195, 2709–2718 (2023). https://doi.org/10.1007/s12010-022-04162-6

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  • DOI: https://doi.org/10.1007/s12010-022-04162-6

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