Acta Veterinaria et Zootechnica Sinica ›› 2025, Vol. 56 ›› Issue (2): 839-850.doi: 10.11843/j.issn.0366-6964.2025.02.032

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Prokaryotic Expression of S1 Protein in Porcine Epidemic Diarrhea Virus and Screening of Its Aptamers

ZHANG Dongxuan(), WANG Zhihao, QIAO Yan, ZHAO Xiaoxiao, FAN Songjie, ZHANG Chao*()   

  1. Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China
  • Received:2024-03-13 Online:2025-02-23 Published:2025-02-26
  • Contact: ZHANG Chao E-mail:15138163667@163.com;chaozhang@henau.edu.cn

Abstract:

This study aimed to express the C-terminal binding domain (S1-CTD) of the porcine epidemic diarrhea virus (PEDV) S1 protein in a soluble prokaryotic system and to screen DNA aptamers targeting S1-CTD using the systematic evolution of ligands by exponential enrichment (SELEX) technique, laying the foundation for the development of therapeutic drugs and detection methods for the PEDV. The PEDV-S1-CTD plasmid was cloned into prokaryotic expression vectors containing maltose-binding protein (MBP), small ubiquitin-like modifier (SUMO), transcription termination factor (NusA), and glutathione S-transferase (GST) tags. Recombinant plasmids pET21b-MBP-S1, pET21b-SUMO-S1, pET21b-NusA-S1, and pET21b-GST-S1 were constructed and transformed into E. coli BL21 for expression. The rMBP-S1 recombinant protein with good solubility and high expression was selected for further experiments. Western blot and indirect immunofluorescence assay (IFA) confirmed the correct expression and receptor binding of the protein. Aptamer Apt-S1-3 and Apt-S1-11 were enriched with the highest affinity (18% and 16.2%, respectively) and specificity, respectively. ELASA determined a dissociation constant of 2.09 nmol for Apt-S1-3, which showed no cross-reactivity with rMBP, rMBP-gD, rMBP-GP5, or BSA proteins. Mfold analysis and molecular docking simulations revealed the aptamer's ability to form stable complexes with the target protein. IFA and flow cytometry confirmed the aptamer's recognition and inhibition of PEDV infection. This study successfully screened a DNA aptamer that binds to the PEDV S1-CTD protein with high affinity and specificity, inhibiting virus infection and providing a foundation for targeted therapy and detection methods for PEDV.

Key words: porcine epidemic diarrhea virus, S1-CTD protein, magnetic bead method, aptamer, affinity

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