鼠伤寒沙门菌鞭毛蛋白FliC生物信息学分析及其对小鼠巨噬细胞的作用

doi:10.16431/j.cnki.1671-7236.2025.08.030

摘要/Abstract

摘要： 【目的】研究鼠伤寒沙门菌FliC蛋白的序列特征、原核表达情况及其对巨噬细胞RAW264.7增殖及细胞因子分泌的影响。【方法】参照GenBank公布的鼠伤寒沙门菌ATCC 14028基因序列设计特异性引物，以鼠伤寒沙门菌ATCC 14028基因组DNA为模板，通过PCR扩增FliC基因CDS序列，应用在线软件分析FliC蛋白结构特征。将FliC基因克隆至原核表达载体pET-28a(+)，将鉴定正确的重组质粒pET-28a-FliC转化大肠杆菌BL21(DE3)感受态细胞，构建重组菌株BL21(pET-28a-FliC)，使用IPTG对重组菌株进行诱导表达并优化重组蛋白的表达条件，对重组蛋白进行镍离子亲和层析纯化，通过SDS-PAGE及Western blotting检测纯化的蛋白。将纯化的重组蛋白与巨噬细胞RAW264.7共同孵育，通过细胞毒性试验检测巨噬细胞增殖情况，采用ELISA法检测巨噬细胞细胞因子分泌情况。【结果】 PCR扩增获得大小为1 488 bp的FliC基因，FliC蛋白分子式为C₂₁₉₄H₃₅₉₇N₆₄₀O₆₄₁S₃，由495个氨基酸组成，分子质量约为52 ku；该蛋白属酸性蛋白，氨基酸残基丙氨酸(12.3%)、苏氨酸(11.5%)出现的频率较高；蛋白为亲水性蛋白，不含信号肽，无跨膜区，包含67个磷酸化位点。二级结构由α-螺旋、β-折叠、β-转角及无规则卷曲构成，占比分别为39.80%、20.20%、4.24%和35.76%。SDS-PAGE及Western blotting检测结果显示，重组蛋白部分可溶，与小鼠抗鞭毛血清有较好的反应原性。50和400 ng/mL重组纯化蛋白能有效刺激巨噬细胞的增殖，在24 h增殖率达到最高，同时可有效刺激巨噬细胞分泌细胞因子白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)，在12 h时细胞因子分泌量最高。【结论】本研究成功扩增得到鼠伤寒沙门菌FliC基因，纯化获得的高纯度FliC蛋白为酸性、亲水性蛋白，该蛋白可促进巨噬细胞增殖及分泌细胞因子，为进一步研究沙门菌FliC蛋白的功能奠定基础。

关键词: 鼠伤寒沙门菌; 鞭毛蛋白; 原核表达; 巨噬细胞; 生物信息学分析

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

中图分类号:

S852.61

张明亮, 王佩, 马磊, 姚畅, 庞蓉, 翁少亭, 马圣明, 连凯琪. 鼠伤寒沙门菌鞭毛蛋白FliC生物信息学分析及其对小鼠巨噬细胞的作用[J]. 中国畜牧兽医, 2025, 52(8): 3820-3829.

ZHANG Mingliang, WANG Pei, MA Lei, YAO Chang, PANG Rong, WENG Shaoting, MA Shengming, LIAN Kaiqi. Bioinformatics Analysis of Flagellar Protein FliC of Salmonella Typhimurium and Its Effect on Mouse Macrophages[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3820-3829.

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