China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (10): 4894-4903.doi: 10.16431/j.cnki.1671-7236.2025.10.034

• Preventive Veterinary Medicine • Previous Articles    

Bioinformatics Analysis of Fusion Protein of Escherichia coli FliC Protein D0/D1 Domain and Pal Protein and Prokaryotic Expression

CHEN Hong1, CHE Yegui1, CHEN Changchun1, DING Xiaoli1, QIU Shulei1, LU Hui1, WANG Yongjuan1, WEN Hongwei1, ZHOU Zixiang2   

  1. 1. Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, China;
    2. Animal Husbandry and Veterinary Station of Liancheng Street, Huai'an 223400, China
  • Revised:2025-03-31 Published:2025-09-30

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Abstract: 【Objective】 The purpose of this study was to construct and express the fusion protein of Escherichia coli (E.coli) FliC protein D0/D1 domain and Pal protein,provide a basis for developing a novel avian enteropathogenic E.coli (APEC) vaccine.【Method】 The amino acid sequence characteristics of FliC and Pal proteins of E.coli O1 serotype strains in GenBank database were analyzed.The D0/D1 domain of FliC protein (N-terminal 5-140 amino acids and C-terminal 499-583 amino acids) and the main structure of Pal protein (21-173 amino acids) were selected to construct the "sandwich-type" fusion protein.The physicochemical properties,secondary structure,tertiary structure,antigenicity,sensitization,linear and discontinuous epitopes of the fusion protein were predicted.The binding ability and immune effect of the fusion protein with immune receptors were evaluated through molecular docking and immune simulation.The base sequence of the fusion protein was synthesized and the vector was constructed using pET-28a(+).The fusion protein was expressed using E.coli BL21(DE3) competent cells.The fusion protein was purified using nickel-agarose gel 6FF and renatured.The reactivity of the fusion protein was verified by Western blotting.【Result】 The molecular mass of the fusion protein was precisely determined to be 42.72 ku,and it was identified as a hydrophilic protein.The composition of the protein’s secondary structure was precisely quantified,with the alpha helix,extended strand,beta turn and random coil accounting for 48.79%,14.25%,6.76% and 30.19%,respectively.Notably,the alpha helix was mainly located within the D0/D1 domain of the FliC protein.The Ramachandran plot of the tertiary structure revealed that the advantage region encompassed an impressive 96.9% of the residues.The fusion protein had outstanding antigenicity,no sensitization,and contained 8 linear epitopes and 5 discontinuous epitopes.Additionally,the fusion protein demonstrated the ability to molecularly dock with Toll-like receptors 2,4 and 5,and the immune simulation results exhibited promising and significant outcomes.The chimeric gene FliCN-Pal-FliCC positive monoclonal was identified by PCR and sequencing and transformed into E.coli BL21 (DE3) competent cells.The fusion protein FliCN-Pal-FliCC was successfully expressed and purified.The fusion protein could react with the serum of E.coli O1 serotype-positive mice.【Conclusion】 In this study,"sandwich" fusion protein FliCN-Pal-FliCC based on FliC and Pal proteins of APEC O1 serotype was successfully constructed and expressed,which provided a research basis for the research and development of candidate antigens of APEC vaccine.

Key words: Escherichia coli; FliC protein; domain; Pal protein; fusion protein; bioinformatics analysis

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