羊布鲁菌外膜囊泡蛋白质组学分析及免疫原性评价

doi:10.11843/j.issn.0366-6964.2025.07.030

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (7): 3378-3389.doi: 10.11843/j.issn.0366-6964.2025.07.030

羊布鲁菌外膜囊泡蛋白质组学分析及免疫原性评价

刘雨欣¹(), 陈思¹(), 高阳¹, 顾德媛¹, 彭海涛¹, 张东¹, 张如¹, 许会会¹, 刘亚乔²^,*(), 杨艳玲¹^,*()

1. 中国农业科学院特产研究所, 长春 130000
2. 中华人民共和国大窑湾海关, 大连 116600

收稿日期:2024-08-28 出版日期:2025-07-23 发布日期:2025-07-25
通讯作者: 刘亚乔,杨艳玲 E-mail:2867366057@qq.com;chensi_1024@163.com;chang375479555@126.com;m18043213639@163.com
作者简介:刘雨欣(2000-)，女，河南邓州人，硕士生，主要从事预防兽医学研究，E-mail: 2867366057@qq.com
陈思(1988-)，辽宁抚顺人，博士，主要从事预防兽医学研究，E-mail：chensi_1024@163.com
基金资助:
“十四五”重点研发计划(2023YFD1800700；2023YFD1800703)；中国农业科学院科技创新工程(CAAS-ZDRW202410)；吉林省肉牛专项(YDZJ202203CGZH051)

Proteomic Analysis and Immunogenicity Evaluation of Outer Membrane Vesicles of Brucella melitensis

LIU Yuxin¹(), CHEN Si¹(), GAO Yang¹, GU Deyuan¹, PENG Haitao¹, ZHANG Dong¹, ZHANG Ru¹, XU Huihui¹, LIU Yaqiao²^,*(), YANG Yanling¹^,*()

1. Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun 130000, China
2. Dayaowan Customs District of P. R. China, Dalian 116600, China

Received:2024-08-28 Online:2025-07-23 Published:2025-07-25
Contact: LIU Yaqiao, YANG Yanling E-mail:2867366057@qq.com;chensi_1024@163.com;chang375479555@126.com;m18043213639@163.com

摘要/Abstract

摘要：

旨在提取羊布鲁菌生物3型外膜囊泡(outer membrane vesicles, OMVs)并分析其蛋白组成成分，制备OMVs亚单位疫苗，评价OMVs疫苗的免疫原性。本研究利用差速离心法提取了OMVs，通过SDS-PAGE、透射电镜和ZeTA纳米粒径对OMVs进行鉴定；利用Label free液相质谱技术鉴定OMVs蛋白质组成成分，并进行GO聚类分析和KEGG富集分析；OMVs亚单位疫苗免疫小鼠后，对小鼠脾脏进行病理检测和T细胞亚群分类检测；对小鼠血清进行IL-2、IFN-γ、TNF-α、IL-4、IL-6、IL-10、IL-17A炎性细胞因子水平检测和IgG 1、IgG 2a和IgG 2b免疫球蛋白含量检测，评价OMVs免疫后小鼠的病理变化和细胞免疫水平。结果显示：成功提取了OMVs，其粒径大小为20~200 nm，相对分子质量在10~130 ku之间。蛋白质组学鉴定结果显示，OMVs中含有1 612种蛋白质，其中包括Omp2b、Omp25、Omp31、Omp19和Bp26等多种外膜蛋白，以及脂蛋白和ABC转运体相关蛋白质。OMVs亚单位疫苗免疫小鼠后，小鼠脾脏未见明显的淋巴细胞排列疏松及坏死；诱导小鼠产生高水平IgG免疫球蛋白含量；同时诱导小鼠血清中IFN-γ、IL-2、IL-6、IL-17A和TNF-α炎性细胞因子表达量上升，IL-10和IL-4炎性细胞因子表达量下降；CD4⁺/CD8⁺T细胞比值增加。本研究证明OMVs中含有多个有效的免疫原性抗原，能诱导小鼠产生较好的细胞免疫水平，为明确布鲁菌OMVs蛋白成分和布鲁菌亚单位疫苗研发奠定了理论基础。

关键词: 布鲁菌, 外膜囊泡, 蛋白质组学, 免疫原性

Abstract:

This study aims to extract Brucella melitensis biotype 3 outer membrane vesicles (OMVs), analyze their protein components, and prepare an OMV subunit vaccine to evaluate its immunogenicity. OMVs were extracted using differential centrifugation, and characterized by SDS-PAGE, transmission electron microscopy, and ZeTA nanoparticle size analysis. The protein components of the OMVs were identified using label-free liquid chromatography-mass spectrometry (LC-MS), followed by Gene Ontology (GO) clustering and KEGG pathway enrichment analysis. After immunizing mice with the OMV subunit vaccine, histopathological analysis of mouse spleens and T cell subpopulation classification were performed. Inflammatory cytokine levels, including IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-10, and IL-17A, as well as immunoglobulin levels (IgG1, IgG2a, and IgG2b) in mouse serum samples, were measured to assess the pathological changes and cellular immune response post-immunization. Results were as follows: OMVs were successfully extracted with particle sizes ranging from 20 to 200 nm and molecular weights between 10 to 130 ku. Proteomic analysis identified 1 612 proteins in OMVs, including several outer membrane proteins such as Omp2b, Omp25, Omp31, Omp19, and Bp26, as well as lipoproteins and proteins related to ABC transporters. After immunization with the OMV subunit vaccine, no significant lymphocyte disorganization or necrosis was observed in mouse spleens. The vaccine induced high levels of IgG immunoglobulin and elevated the expression of inflammatory cytokines IFN-γ, IL-2, IL-6, IL-17A, and TNF-α, while reducing IL-10 and IL-4 levels. The CD4⁺/CD8⁺T cell ratio also increased. The study demonstrates that OMVs contain several effective immunogenic antigens capable of inducing strong cellular immune responses in mice. This lays the theoretical foundation for identifying the protein components of OMVs and developing Brucella subunit vaccines.

Key words: Brucella, outer membrane vesicles, proteomics, immunogenicity

中图分类号:

S852.614

刘雨欣, 陈思, 高阳, 顾德媛, 彭海涛, 张东, 张如, 许会会, 刘亚乔, 杨艳玲. 羊布鲁菌外膜囊泡蛋白质组学分析及免疫原性评价[J]. 畜牧兽医学报, 2025, 56(7): 3378-3389.

LIU Yuxin, CHEN Si, GAO Yang, GU Deyuan, PENG Haitao, ZHANG Dong, ZHANG Ru, XU Huihui, LIU Yaqiao, YANG Yanling. Proteomic Analysis and Immunogenicity Evaluation of Outer Membrane Vesicles of Brucella melitensis[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3378-3389.

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参考文献 39

1	MORENO E . Retrospective and prospective perspectives on zoonotic brucellosis[J]. Front Microbiol, 2014, 5, 213.
2	HE C Y , YANG J H , YE Y B , et al. Proteomic and antibody profiles reveal antigenic composition and signatures of bacterial ghost vaccine of Brucella abortus A19[J]. Front Immunol, 2022, 13, 874871.
3	GALIŃSKAó E M , ZAGÓRSKI J . Brucellosis in humans——etiology, diagnostics, clinical forms[J]. Ann Agric Environ Med, 2013, 20 (2): 233- 238.
4	敖敏高娃, 张国庆, 郑旭, 等. 呼伦贝尔地区人感染布鲁氏菌菌株生物学特性分析[J]. 疾病监测与控制, 2019, 13 (1): 1-2, 4.
	AO M G W , ZHANG G Q , ZHENG X , et al. Analysis of biological characteristics of brucella isolated from infected human in hulunbeier[J]. Journal of Diseases Monitor & Control, 2019, 13 (1): 1-2, 4.
5	黄天鹏, 郭旭, 孙长云, 等. 呼伦贝尔地区牛体表寄生蜱中羊种布鲁氏菌的分离与鉴定[J]. 中国农业科学, 2022, 55 (2): 415- 424.
	HUANG T P , GUO X , SUN C Y , et al. Isolation and identification of Brucella melitensis from ticks on cattle surface in hulunbuir area[J]. Scientia Agricultura Sinica, 2022, 55 (2): 415- 424.
6	MATHUR S , BANAI M , COHEN D . Natural Brucella melitensis infection and rev. 1 vaccination induce specific Brucella O-polysaccharide antibodies involved in complement mediated Brucella cell killing[J]. Vaccines (Basel), 2022, 10 (2): 317.
7	LI Z Q , SHI J X , FU W D , et al. A Brucella melitensis M5-90 wboA deletion strain is attenuated and enhances vaccine efficacy[J]. Mol Immunol, 2015, 66 (2): 276- 283.
8	HOU H H , LIU X F , PENG Q S . The advances in brucellosis vaccines[J]. Vaccine, 2019, 37 (30): 3981- 3988.
9	胡森, 郑孝辉, 王加兰, 等. 马耳它布氏杆菌bp26基因缺失株的构建及鉴定[J]. 中国预防兽医学报, 2009, 31 (8): 583- 586.
	HU S , ZHENG X H , WANG J L , et al. Development of a mutant Brucella Melitensis as vaccine in mice[J]. Chinese Journal of Preventive Veterinary Medicine, 2009, 31 (8): 583- 586.
10	CASTAÑO-ZUBIETA M R , ROSSETTI C A , GARCÍA-GONZÁLEZ D G , et al. Evaluation of the safety profile of the vaccine candidate Brucella melitensis 16MΔvjbR strain in goats[J]. Vaccine, 2021, 39 (3): 617- 625.
11	DEATHERAGE B L , COOKSON B T . Membrane vesicle release in bacteria, eukaryotes, and archaea: a conserved yet underappreciated aspect of microbial life[J]. Infect Immun, 2012, 80 (6): 1948- 1957.
12	VANAJA S K , RUSSO A J , BEHL B , et al. Bacterial outer membrane vesicles mediate cytosolic localization of LPS and caspase-11 activation[J]. Cell, 2016, 165 (5): 1106- 1119.
13	KOEPPEN K , HAMPTON T H , JAREK M , et al. A novel mechanism of host-pathogen interaction through sRNA in bacterial outer membrane vesicles[J]. PLoS Pathog, 2016, 12 (6): e1005672.
14	KESAVAN D , VASUDEVAN A , WU L , et al. Integrative analysis of outer membrane vesicles proteomics and whole-cell transcriptome analysis of eravacycline induced Acinetobacter baumannii strains[J]. BMC Microbiol, 2020, 20 (1): 31.
15	BHAR S , EDELMANN M J , JONES M K . Characterization and proteomic analysis of outer membrane vesicles from a commensal microbe, Enterobacter cloacae[J]. J Proteomics, 2021, 231, 103994.
16	KUEHN M J , KESTY N C . Bacterial outer membrane vesicles and the host-pathogen interaction[J]. Genes Dev, 2005, 19 (22): 2645- 2655.
17	XIONG X , LI B W , ZHOU Z X , et al. The VirB system plays a crucial role in Brucella intracellular infection[J]. Int J Mol Sci, 2021, 22 (24): 13637.
18	ROOP Ⅱ R M , BARTON I S , HOPERSBERGER D , et al. Uncovering the hidden credentials of Brucella virulence[J]. Microbiol Mol Biol Rev, 2021, 85 (1): e00021- 19.
19	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.
20	ALAIDAROUS M , VE T , CASEY L W , et al. Mechanism of bacterial interference with TLR4 signaling by Brucella Toll/interleukin-1 receptor domain-containing protein TcpB[J]. J Biol Chem, 2014, 289 (2): 654- 668.
21	GAMAZO C , WINTER A J , MORIYÓN I , et al. Comparative analyses of proteins extracted by hot saline or released spontaneously into outer membrane blebs from field strains of Brucella ovis and Brucella melitensis[J]. Infect Immun, 1989, 57 (5): 1419- 1426.
22	AVILA-CALDERÓN E D , MEDINA-CHÁVEZ O , FLORES-ROMO L , et al. Outer membrane vesicles from Brucella melitensis modulate immune response and induce cytoskeleton rearrangement in peripheral blood mononuclear cells[J]. Front Microbiol, 2020, 11, 556795.
23	KNOX K W , VESK M , WORK E . Relation between excreted lipopolysaccharide complexes and surface structures of a lysine-limited culture of Escherichia coli[J]. J Bacteriol, 1966, 92 (4): 1206- 1217.
24	AVILA-CALDERÓN E D , LOPEZ-MERINO A , JAIN N , et al. Characterization of outer membrane vesicles from Brucella melitensis and protection induced in mice[J]. Clin Dev Immunol, 2012, 2012, 352493.
25	KAUR G , SINGH S , KUMAR B V S , et al. Characterization and immunogenicity of outer membrane vesicles from Brucella abortus[J]. J Immunoassay Immunochem, 2016, 37 (3): 261- 272.
26	曲俊泽, 陈天华, 姚明东, 等. ABC转运蛋白及其在合成生物学中的应用[J]. 生物工程学报, 2020, 36 (9): 1754- 1766.
	QU J Z , CHEN T H , YAO M D , et al. ABC transporter and its application in synthetic biology[J]. Chinese Journal of Biotechnology, 2020, 36 (9): 1754- 1766.
27	ZHANG K , WANG H , GUO F , et al. OMP31 of Brucella melitensis 16M impairs the apoptosis of macrophages triggered by TNF-α[J]. Exp Ther Med, 2016, 12 (4): 2783- 2789.
28	CLOECKAERT A , TIBOR A , ZYGMUNT M S . Brucella outer membrane lipoproteins share antigenic determinants with bacteria of the family Rhizobiaceae[J]. Clin Diagn Lab Immunol, 1999, 6 (4): 627- 629.
29	BOWDEN R A , ESTEIN S M , ZYGMUNT M S , et al. Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies[J]. Microbes Infect, 2000, 2 (5): 481- 488.
30	李大伟, 朱玥洁, 霍怡杉, 等. 布鲁氏菌Omp2b优势T-B联合抗原表位的免疫应答特点研究[J]. 中国人兽共患病学报, 2021, 37 (8): 683- 687.
	LI D W , ZHU Y J , HUO Y S , et al. Study of the immune response characteristics of Brucella Omp2b dominant T-B combined antigen epitope[J]. Chinese Journal of Zoonoses, 2021, 37 (8): 683- 687.
31	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.
32	LI R Z , LIU W L , YIN X R , et al. Brucella spp. Omp25 promotes proteasome-mediated cGAS degradation to attenuate IFN-β production[J]. Front Microbiol, 2021, 12, 702881.
33	LUO X M , ZHANG X J , WU X C , et al. Brucella downregulates tumor necrosis factor-α to promote intracellular survival via omp25 regulation of different MicroRNAs in porcine and murine macrophages[J]. Front Immunol, 2018, 8, 2013.
34	VERDIGUEL-FERNÁNDEZ L , OROPEZA-NAVARRO R , BASURTO-ALCÁNTARA F J , et al. Omp31 plays an important role on outer membrane properties and intracellular survival of Brucella melitensis in murine macrophages and HeLa cells[J]. Arch Microbiol, 2017, 199 (7): 971- 978.
35	AZIZPOUR MAGHVAN M , JAFARI P , HOSEINI S D , et al. Cloning and expression of B. mellitensis bp26 gene in Lactococcus lactis as a food grade vaccine[J]. Avicenna J Med Biotechnol, 2019, 11 (3): 264- 267.
36	KIM D H , SON B G , LIM J J , et al. The role of a Brucella abortus lipoprotein in intracellular replication and pathogenicity in experimentally infected mice[J]. Microb Pathog, 2013, 54, 34- 39.
37	RAHMANPOUR M , KERAMAT F , JOURGHASEMI S , et al. Direct correlation between Th1 and Th17 responses in immunity to Brucella infection[J]. Microb Infect, 2019, 21 (10): 441- 448.
38	GOENKA R , PARENT M A , ELZER P H , et al. B cell-deficient mice display markedly enhanced resistance to the intracellular bacterium Brucella abortus[J]. J Infect Dis, 2011, 203 (8): 1136- 1146.
39	ZHAN Y , LIU Z , CHEERS C . Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms[J]. Infect Immun, 1996, 64 (7): 2782- 2786.

序号Number	蛋白种类Protein Accession	蛋白描述Protein description	基因名称Gene name
1	A0A0F6AQQ4	DUF2059 domain-containing protein	BAbS19_I09550
2	C0G894	Peptidoglycan-associated protein	pal
3	A0A011T368	Invasion associated locus B family protein	F9K89_03010
4	A0A0E1WYK9	Extracellular solute-binding protein	BALG_02442
5	U4VI36	Thiosulfate transporter subunit	Q644_16500
6	A0A0E1WY67	Tol-Pal system protein TolB	tolB
7	A0A0E1X5J8	Penicillin amidase	BALG_01689
8	A0A0E1WY23	Cell division coordinator CpoB	cpoB
9	A0A0E1WZN5	Sulfate ABC transporter	BALG_00414
10	C4IVQ1	Peptide ABC transporter, periplasmic peptide-binding protein	BAAA_7000669
11	A0A7L7MQK5	Nitrous-oxide reductase	nosZ
12	A0A0E1X5F6	Outer membrane protein assembly factor BamD	bamD
13	A0A0E1X039	Lectin-like protein BA14k	BALG_00492
14	A0AA87DN68	Outer membrane antigenic lipoprotein B	BIBO2_0180
15	D7GYV1	Outer membrane protein	BAYG_01468
16	P0A3Z5	Probable periplasmic serine endoprotease DegP-like	htrA
17	A0A0F6ATR6	Zinc protease	BAbS19_II01900
18	A0A7U8KGZ0	Uncharacterized protein	BAFG_02971
19	A0A0E1XAF7	Signal peptide protein	BALG_00361
20	A0AA86ZXD2	Lipoprotein	BIBO2_2383

项目Items	组别Groups
项目Items	PBS	OMVs
CD4⁺	2 310	2 004.67
CD8⁺	374	283.67
CD4⁺/CD8⁺	6.18^a	7.07^a

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羊布鲁菌外膜囊泡蛋白质组学分析及免疫原性评价

Proteomic Analysis and Immunogenicity Evaluation of Outer Membrane Vesicles of Brucella melitensis

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