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

doi:10.16431/j.cnki.1671-7236.2024.05.006

摘要/Abstract

摘要： 【目的】对布鲁氏菌BspF蛋白进行生物信息学分析,并进一步探究BspF蛋白对巨噬细胞炎症因子表达的影响。【方法】利用生物信息学在线软件分析布鲁氏菌BspF蛋白的理化性质、亲/疏水性、信号肽、跨膜区、抗原决定簇及结构域、二级结构、三级结构。构建重组质粒pET-32a-BspF并将其转化大肠杆菌BL21(DE3)感受态细胞,用IPTG诱导表达BspF蛋白并进行纯化,通过SDS-PAGE和Western blotting检测蛋白表达及纯化情况。通过CCK-8法检测纯化后BspF蛋白对小鼠巨噬细胞(RAW264.7)的毒性,利用实时荧光定量PCR和Western blotting分别检测BspF蛋白对细胞中炎症相关因子mRNA和蛋白表达的影响。【结果】BspF蛋白分子质量为48.75 ku,属于不稳定蛋白,具有15个抗原决定簇,不存在跨膜区域和信号肽,二级结构以α-螺旋为主。成功构建了布鲁氏菌BspF基因原核表达载体,诱导表达后获得大小为60 ku的目标蛋白。纯化后蛋白以50 μg/mL终浓度刺激RAW264.7细胞24 h后,与对照组相比,BspF组细胞中NLRP3、Caspase-1、IL-1β、IL-18的mRNA表达量均显著或极显著升高(P＜0.05;P＜0.01),NLRP3、Pro-Caspase-1蛋白表达量均极显著升高(P＜0.01)。【结论】布鲁氏菌BspF蛋白能通过NLRP3炎症小体触发宿主细胞炎症反应,促进巨噬细胞炎症因子的表达。结果为布鲁氏菌致炎机制的研究和抗炎靶点的筛选提供理论基础。

关键词: 布鲁氏菌; BspF蛋白; 生物信息学; 原核表达; 炎性因子

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.

Key words: Brucella; BspF protein; bioinformatics; prokaryotic expression; inflammatory

中图分类号:

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.

SUN Can, WEI Shuo, YAN Yujing, ZHAO Tianyi, ZHU Dexin, DENG Xingmei, GUO Jia, ZHANG Hui, SUN Zhihua. Bioinformatics Analysis of Brucella BspF Protein and Its Effect on Expression of Inflammatory Factors in Macrophages[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 1846-1856.

参考文献

[1] 徐朕宇,徐锦凤,邓肖玉,等.布鲁氏菌外膜囊泡生物信息学分析及免疫原性评价[J].中国畜牧兽医,2023,50(7):2906-2914. XU Z Y,XU J F,DENG X Y,et al.Bioinformatics analysis and immunogenicity evaluation of Brucella outer membrane vesicles[J].China Animal Husbandry & Veterinary Medicine,2023,50(7):2906-2914.(in Chinese)
[2] 文志,韩艳秋,王俊瑞.布鲁氏菌毒力因子研究进展[J].微生物学通报,2021,48(3):842-848. WEN Z,HAN Y Q,WANG J R.Virulence factors of Brucella:A review[J].Microbiology China,2021,48(3):842-848.(in Chinese)
[3] 袁莎,陈鹏博,陈创夫.布鲁氏菌LPS对小鼠巨噬细胞中NLRP3炎症小体转录水平的影响[J].中国畜牧兽医,2015,42(1):9-16. YUAN S,CHEN P B,CHEN C F.Effect of Brucella LPS on NLRP3 inflammasome transcriptional level of mouse macrophages[J].China Animal Husbandry & Veterinary Medicine,2015,42(1):9-16.(in Chinese)
[4] 陶婷婷,赵天艺,李佳,等.TGF-β1在布鲁氏菌致炎过程中的作用及其与NLRP3炎症小体的相关性研究[J].动物医学进展,2022,43(2):48-53. TAO T T,ZHAO T Y,LI J,et al.Study on the role of TGF-β1 in Brucella induce inflammation and its correlation with NLRP3 inflammasome[J].Progress in Veterinary Medicine,2022,43(2):48-53.(in Chinese)
[5] 朱德馨,吴洁,郭嘉,等.布鲁氏菌BAB-RS25305蛋白对细胞炎症因子表达的影响[J].动物医学进展,2021,42(9):1-6. ZHU D X,WU J,GUO J,et al.Effect of BAB-RS25305 protein on expression of inflammatory cytokines[J].Progress in Veterinary Medicine,2021,42(9):1-6.(in Chinese)
[6] MILLER C N,SMITH E P,CUNDIFF J A,et al.A Brucella type Ⅳ effector targets the COG tethering complex to remodel host secretory traffic and promote intracellular replication[J].Cell Host & Microbe,2017,22:317-329.
[7] 邱润辉,关飞虎,王梓行,等.布鲁氏菌分泌蛋白BMCO基因缺失株的构建及生物学特性研究[J].中国畜牧兽医,2022,49(5):1630-1640. QIU R H,GUAN F H,WANG Z H,et al.Construction and biological characteristics of Brucella secrering protein BMCO gene deletion strain[J].China Animal Husbandry & Veterinary Medicine,2022,49(5):1630-1640.(in Chinese)
[8] BORGHESAN E,SMITH E P,MYENI A,et al.A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication[J].The European Molecular Biology Organization Journal,2021,40(19):e107664.
[9] LIN R,LI A,LI Y,et al.The Brucella effector protein BspF regulates apoptosis through the crotonylation of p53[J].Microorganisms,2023,11(9):2322.
[10] 马忠臣,胡瑞瑞,李芮芮,等.布鲁氏菌的胞内生存机制研究进展[J].动物医学进展,2021,42(6):71-78. MA Z C,HU R R,LI R R,et al.Progress on intracellular surivival mechanism of Brucella[J].Progress in Veterinary Medincine,2021,42(6):71-78.(in Chinese)
[11] ZHOU Y,BU Z,QIAN J,et al.The UTP-glucose-1-phosphate uridylyltransferase of Brucella melitensis inhibits the activation of NF-κB via regulating the bacterial type Ⅳ secretion system[J].International Journal of Biological Macromolecules,2020,164:3098-3104.
[12] XIONG X,LI B,ZHOU Z,et al.The VirB system plays a crucial role in Brucella intracellular infection[J].International Journal of Molecular Sciences,2021,22(24):13637.
[13] LUNDQUIST K,BILLINGS E,BI M,et al.The assembly of β-barrel membrane proteins by BAM and SAM[J].Molecular Microbiology,2021,115(3):425-435.
[14] 王书利,李梦含,高萌萌,等.布鲁氏菌分泌蛋白BPE043的功能及其对细胞凋亡基因表达的影响[J/OL].中国动物传染病学报,2022.Doi:10.19958/j.cnki.cn31-2031/s.20221207.003. WANG S L,LI M H,GAO M M,et al.Functional and effect on expressions of apoptosis-associated genes of Brucella type Ⅳ secretion system effector protein BPE043[J].Chinese Journal of Animal Infectious Diseases,2022.Doi:10.19958/j.cnki.cn31-2031/s.20221207.003.(in Chinese)
[15] 陈蕾,张广冻,李俊玫,等.布鲁氏菌入侵相关基因IalB缺失株的构建及生物学特性分析[J].微生物学报,2023,63(3):1254-1268. CHEN L,ZHANG G D,LI J M,et al.Construction and characterization of IalB-deleted strain of Brucella suis[J].Acta Microbiologica Sinica,2023,63(3):1254-1268.(in Chinese)
[16] 杨江流,乔军伟,宋青山,等.布鲁氏菌S19毒力基因BvfA的原核表达及生物信息学分析[J].中国畜牧兽医,2021,48(8):2746-2753. YANG J L,QIAO J W,SONG Q S,et al.Prokaryotic expression and bioinformatics analysis of virulence gene BvfA of Brucella S19[J].China Animal Husbandry & Veterinary Medicine, 2021,48(8):2746-2753.(in Chinese)
[17] 肖洋洋,马忠臣,李芮芮,等.布鲁氏菌分泌蛋白BspI生物信息学分析及多克隆抗体制备[J].中国畜牧兽医,2022,49(8):3112-3121. XIAO Y Y,MA Z C,LI R R,et al.Bioinformatic analysis and polyclonal preparation of Brucella secreted protein BspI[J].China Animal Husbandry & Veterinary Medicine,2022,49(8):3112-3121.(in Chinese)
[18] 蒋丹露,刘阳阳,孙如愚,等.NLRP3炎症小体介导的炎症相关疾病研究进展[J].生命科学,2017,29(9):898-907. JIANG D L,LIU Y Y,SUN R Y,et al.NLRP3 inflamamasome and its role in inflammation related diseases[J].Chinese Bulletin of Life Sciences,2017,29(9):898-907.(in Chinese)
[19] JO E K,KIM J K,SHIN D M D,et al.Molecular mechanisms regulating NLRP3 inflammasome activation[J].Cellular & Molecular Immunology,2016,13(2):148-159.
[20] BRONNER D N,ABUAITA B H,CHEN X,et al.Endoplasmic reticulum stress activates the inflammasome via NLRP3-Caspase-2 driven mitochondrial damage[J].Immunity,2015,43:451-462.
[21] LOB S,AMANN N,KUHN C,et al.Interleukin-1 beta is significantly upregulated in the decidua of spontaneous and recurrent miscarriage placentas[J].Journal of Reproductive Immunology,2021,144:103283.