中国畜牧兽医2024，Vol.51Issue(5)：1846-1856,11.DOI:10.16431/j.cnki.1671-7236.2024.05.006
布鲁氏菌BspF蛋白生物信息学分析及其对巨噬细胞炎症因子表达的影响
Bioinformatics Analysis of Brucella BspF Protein and Its Effect on Expression of Inflammatory Factors in Macrophages
摘要
Abstract
[Objective]The aim of this study was to analyze the bioinformatics of Brucella BspF protein,and further explore the effect of BspF protein on the expression of inflammatory cytokines in macrophages.[Method]The physical and chemical properties,hydrophilicity and hydrophobicity,signal peptide,transmembrane region,antigenic determinant and domain,secondary structure and tertiary structure of Brucella BspF protein were analyzed by bioinformatics online software.Constructed recombinant plasmid pET-32a-BspF and then transformed into Escherichia coli BL21(DE3)competent cells.BspF protein was induced by IPTG and purified.The expression and purification of BSPF protein were detected by SDS-PAGE and Western blotting.The toxicity of purified BspF protein on mouse macrophages(RAW264.7)was detected by CCK-8 method,and the effects of BspF protein on mRNA and protein expression of inflammatory factors in cells were detected by Real-time quantitative PCR and Western blotting,respectively.[Result]The results showed that BspF protein,with a molecular mass of 48.75 ku,was an unstable protein with 15 antigenic determinant,no transmembrane region and no signal peptide,and the secondary structure was dominated by alpha helix.The prokaryotic expression vector of Brucella BspF gene was successfully constructed,and the target protein with the size of 60 ku was obtained after induced expression.Compared with control group,after the purified protein was stimulated at the final concentration of 50 μg/mL for 24 h,the mRNA expression of NLRP3,Caspase-1,IL-1β and IL-18 in BspF group were significantly or extremely significantly increased(P＜0.05 or P＜0.01),the protein expression of NLRP3 and Pro-Caspase-1 were extremely significantly increased(P＜0.01).[Conclusion]Brucella BspF protein could trigger the inflammatory response of host cells through the NLRP3 inflammatome and promote the expression of inflammatory factors in macrophages,which provided a theoretical basis for the study of the inflammatogenic mechanism of Brucella and the screening of anti-inflammatory targets.关键词
布鲁氏菌/BspF蛋白/生物信息学/原核表达/炎性因子Key words
Brucella/BspF protein/bioinformatics/prokaryotic expression/inflammatory分类
生物科学引用本文复制引用
孙灿,魏硕,鄢余静,赵天艺,朱德馨,邓兴梅,郭嘉,张辉,孙志华..布鲁氏菌BspF蛋白生物信息学分析及其对巨噬细胞炎症因子表达的影响[J].中国畜牧兽医,2024,51(5):1846-1856,11.基金项目
石河子大学高层次人才项目(RCZK202038) （RCZK202038）
