China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (8): 3820-3829.doi: 10.16431/j.cnki.1671-7236.2025.08.030

• Preventive Veterinary Medicine • Previous Articles    

Bioinformatics Analysis of Flagellar Protein FliC of Salmonella Typhimurium and Its Effect on Mouse Macrophages

ZHANG Mingliang1,2, WANG Pei1,2,3, MA Lei1,2, YAO Chang1, PANG Rong1, WENG Shaoting1,2, MA Shengming1,2, LIAN Kaiqi1,2   

  1. 1. College of Biological Science and Food Engineering, Anyang Institute of Technology, Anyang 455000, China;
    2. Henan Joint International Research Laboratory of Veterinary Biologics Research and Application, Anyang 455000, China;
    3. College of Animal Science and Technology, Henan Institute of Science and Technology, Xinxiang 453000, China
  • Received:2024-12-22 Published:2025-08-02

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Abstract: 【Objective】 This study aimed to investigate the sequence characteristics of Salmonella Typhimurium FliC protein,its prokaryotic expression,and effects on the proliferation and cytokine secretion of macrophage RAW264.7 cells. 【Method】 Specific primers were designed based on Salmonella Typhimurium ATCC 14028 gene sequence from GenBank,and FliC gene coding sequence was amplified by PCR using the Salmonella Typhimurium ATCC 14028 genome DNA as a template.Online bioinformatics tools were used to analyze the structural features of FliC protein.The abtained FliC gene was cloned into the prokaryotic expression vector pET-28a(+),and the recombinant plasmid pET-28a-FliC was transformed into Escherichia coli BL21(DE3) competent cells to construct the recombinant strain BL21 (pET-28a-FliC).The expression of the recombinant protein was induced with IPTG,and the expression conditions were optimized.The recombinant protein was purified by Ni-NTA and verified by SDS-PAGE and Western blotting.The purified recombinant protein was co-incubated with macrophage RAW264.7,then the proliferation of macrophages was detected by cytotoxicity test,and the cytokines secretion of macrophage was detected by ELISA. 【Result】 FliC gene with 1 488 bp was successfully amplified,and FliC protein (molecular formula:C2194H3597N640O641S3) consisted of 495 amino acids with a molecular weight of approximately 52 ku.The protein was acidic,with high frequencies of Ala (12.3%) and Thr (11.5%) residues.It was hydrophilic,lacked a signal peptide and transmembrane domains,and contained 67 phosphorylation sites.The secondary structure of the protein consisted of alpha helix,beta sheet,beta turn and random coil,with the proportions of 39.80%,20.2%,4.24% and 35.76%,respectively.SDS-PAGE and Western blotting showed that the recombinant protein was partially soluble and had a good reactivity with flagellum serum from mice.The recombinant purified protein of 50 and 400 ng/mL could effectively stimulate the proliferation of macrophages,and the proliferation rate reached the highest at 24 h.At the same time,it could effectively stimulate macrophages to secrete cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α),with peak cytokine levels at 12 h. 【Conclusion】 FliC gene of Salmonella was successfully amplified.The high purity FliC protein was acidic,hydrophilic,which could promote the proliferation of macrophages and secretion of cytokines.This study laid a foundation for in-depth analysis of function of the Salmonella FliC protein.

Key words: Salmonella Typhimurium; flagellin protein; prokaryotic expression; macrophage; bioinformatics analysis

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