布鲁氏菌外膜囊泡生物信息学分析及免疫原性评价

doi:10.16431/j.cnki.1671-7236.2023.07.030

Abstract

Abstract: 【Objective】 The purpose of this experiment was to explore the prospects of Brucella outer membrane vesicles (OMVs) for use in subunit vaccines, prepare subunit vaccines of Brucella OMVs, and assess the immunogenicity of Brucella OMVs.【Method】 OMVs of Brucella melitensis were prepared by high-speed centrifugation, and the morphological and fractional analyses of the prepared OMVs were performed by transmission electron microscopy, SDS-PAGE and bioinformatics analysis.Mice were immunized after emulsification of OMVs using nanoadjuvant (LDH) and Freund's adjuvant, and the specific antibodies were detected by indirect ELISA method for OMVs at 7, 14, 21, 28 and 35 d after immunization, and the IgG antibody levels in serum of mice post-immunization were detected using the mouse IgG ELISA kit, so as to assess the level of humoral immunity induced by OMVs.The level of cellular immunity induced by OMVs was assessed by isolation of mouse spleen lymphocytes with cytokine (IL-4 and IFN-γ) assay and flow cytometry assay of T lymphocyte subpopulations in the peripheral blood of mice.【Result】 The OMVs were successfully extracted and purified, with diameters ranging from 20 to 160 nm.Bioinformatics analysis showed that the main constituent proteins of OMVs, OMP25, OMP31, OMP16, OMP19, BP26 and SOD were hydrophilic and antigenic proteins, and there were several B and T cell dominant.Compared with PBS group, after immunization with OMVs, the different adjuvant groups induced high levels of specific and IgG antibodies in mice (P<0.01).Meanwhile, OMVs significantly induced IFN-γ production in mouse spleen lymphocytes (P<0.05), and promoted the expression of CD4⁺ T cells (P<0.05).【Conclusion】 This study confirmed that Brucella OMVs of sheep species had strong immunogenicity and could induce strong humoral and cellular immune responses in mice, laid a theoretical foundation for the development of a new subunit vaccine for Brucella.

Key words: Brucella; outer membrane vesicles (OMVs); immunogenicity

CLC Number:

S852.61^＋4

XU Zhenyu, XU Jinfeng, DENG Xiaoyu, WANG Yueli, HE Jinke, XIE Shanshan, WANG Zhen, YI Jihai, WANG Yong, MIAO Yuhe, CUI Lijin, CHEN Chuangfu. Bioinformatics Analysis and Immunogenicity Evaluation of Brucella Outer Membrane Vesicles[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2906-2914.

References

[1] 杨琴, 邓肖玉, 谢珊珊, 等.牛种布鲁氏菌Ⅳ型分泌系统对巨噬细胞内质网应激和细胞凋亡的影响[J].畜牧兽医学报, 2022, 53(4):1192-1200. YANG Q, DENG X Y, XIE S S, et al.Effects of Brucella bovis type Ⅳ secretion system on endoplasmic reticulum stress and apoptosis of macrophages[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(4):1192-1200.(in Chinese)
[2] 孙浩杰, 徐磊, 孙佳丽, 等.羊种布鲁氏菌WadC基因缺失株的构建与鉴定[J].中国畜牧兽医, 2020, 47(12):3852-3860. SUN H J, XU L, SUN J L, et al.Construction and identification of Brucella WadC gene deletion strain[J].China Animal Husbandry & Veterinary Medicine, 2020, 47(12):3852-3860.(in Chinese)
[3] 穆国冬, 薛智洋, 陆彪, 等.家畜布鲁氏菌病的综合防控措施及疫苗研究进展[J].吉林畜牧兽医, 2021, 42(12):9-10. MU G T, XUE Z Y, LU B, et al.Progress of integrated prevention and control measures and vaccine research of brucellosis in domestic animals[J].Jilin Animal Husbandry and Veterinary Medicine, 2021, 42(12):9-10.(in Chinese)
[4] 付思美.布鲁氏菌保护性抗原的筛选和初步评价[D].长春:吉林大学, 2011. FU S M.Screening and validating the protective protect vaccine for Brucella[D].Changchun:Jilin University, 2011.(in Chinese)
[5] 郭佳怡.布鲁氏菌病疫苗应用现状与防控措施[J].中国畜牧业, 2021, 6:42-43. GUO J Y.Application status of brucellosis vaccine and control measures of brucellosis[J].China Animal Industry, 2021, 6:42-43.(in Chinese)
[6] 孙天浩, 马忠臣, 张欢, 等.家畜布鲁氏菌病相关疫苗的研究进展[J].现代畜牧兽医, 2021, 7:80-86. SUN T H, MA Z C, ZHANG H, et al.Progress of research on brucellosis vaccine for livestock[J].Modern Journal of Animal Husbandry and Veterinary Medicine, 2021, 7:80-86.(in Chinese)
[7] KAPARAKIS-LIASKOS M, FERRERO R L.Immune modulation by bacterial outer membrane vesicles[J].Nature Reviews Immunology, 2015, 15(6):375-387.
[8] 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].Clinical and Developmental Immunology, 2012, 2012:352493.
[9] MICOLI F, MACLENNAN C A.Outer membrane vesicle vaccines[J].Seminars in Immunology, 2020, 50:101433.
[10] BAGHERI NEJAD R, YAHYARAEYAT R, ES-HAGHI A, et al.Induction of specific cell-mediated immune responses and protection in BALB/c mice by vaccination with outer membrane vesicles from a Brucella melitensis human isolate[J].Acta Pathologica, Microbiologica, et Immunologica Scandinavica, 2019, 127(12):797-804.
[11] 易洁, 刘青, 孔庆科.革兰氏阴性菌外膜囊泡作为亚单位疫苗的研究进展[J].微生物学报, 2016, 56(6):911-921. YI J, LIU Q, KONG Q K.Progress in the study of outer membrane vesicles of Gram-negative bacteria as subunit vaccines[J].Acta Microbiologica Sinica, 2016, 56(6):911-921.(in Chinese)
[12] DURAND V, MACKENZIE J, DE LEON J, et al.Role of lipopolysaccharide in the induction of type Ⅰinterferon-dependent cross-priming and IL-10 production in mice by meningococcal outer membrane vesicles[J].Vaccine, 2009, 27(13):1912-1922.
[13] ALANIZ R C, DEATHERAGE B L, LARA J C, et al.Membrane vesicles are immunogenic facsimiles of Salmonella Typhimurium that potently activate dendritic cells, prime B and T cell responses, and stimulate protective immunity in vivo[J].Journal of Immunology, 2007, 179(11):7692-7701.
[14] FINDLOW J, HOLLAND A, ANDREWS N, et al.Comparison of phenotypically indistinguishable but geographically distinct Neisseria meningitidis group B isolates in a serum bactericidal antibody assay[J].Clinical and Vaccine Immunology, 2007, 14(11):1451-1457.
[15] 周舒扬, 张丕奇, 戴肖东, 等.细菌外膜囊泡(OMV)研究进展[J].微生物学杂志, 2021, 41(6):83-89. ZHOU S Y, ZHANG P Q, DAI X D, et al.Advances in bacterial outer membrane vesicles (OMV)[J].Journal of Microbiology, 2021, 41(6):83-89.(in Chinese)
[16] GAMAZO C, MORIYÓN I.Release of outer membrane fragments by exponentially growing Brucella melitensis cells[J].Infection and Immunity, 1987, 55(3):609-615.
[17] SOCORRO RUIZ-PALMA M D, AVILA-CALDERÓN E D, AGUILERA-ARREOLA M G, et al.Comparative proteomic analysis of outer membrane vesicles from Brucella suis, Brucella ovis, Brucella canis and Brucella neotomae[J].Archives of Microbiology, 2021, 203(4):1611-1626.
[18] 李笋.鲍曼不动杆菌外膜蛋白(OMP)和外膜囊泡(OMV)疫苗制备及其免疫保护作用研究[D].重庆:中国人民解放军陆军军医大学, 2020. LI S.Preparation and protective efficacy research of Acinetobacter baumannii outer membrane protein (OMP) and outer membrane vesicles (OMV) vaccine[D].Chongqing:Chinese People's Liberation Army Army Medical University, 2020.(in Chinese)
[19] YAZD M, SADAT S D, KHALFEH-GHOL M, et al.Extraction and biological evaluation of external membrane vesicles of Brucella abortus as a candidate for brucellosis vaccine[J].Journal of Surgery and Medicine, 2020, 4(7):540-544.
[20] RAHMANPOUR M, KERAMAT F, JOURGHASEMI S, et al.Direct correlation between Th1 and Th17 responses in immunity to Brucella infection[J].Microbes and Infection, 2019, 21(10):441-448.
[21] 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].Journal of Infectious Diseases, 2011, 203(8):1136-1146.

Bioinformatics Analysis and Immunogenicity Evaluation of Brucella Outer Membrane Vesicles

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	ZHANG Zhewei, DAI Xiaotong, WEI Zudan, CHEN Yunpeng, GAN Jing, BEI Weicheng. Study on Prokaryotic Expression and Immunogenicity of OmpW and TbpA Proteins in Swine Pasteurella multocida [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1489-1498.
[2]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[3]	WEI Chunyan, GUO Jia, ZHU Dexin, ZHANG Wei, ZHU Jiale, DEN Xingmei, JIA Sifeng, LIU Liangbo, ZHANG Hui. Construction of Deletion Strain of Brucella BPE159 Gene and Effect of BPE159 Protein on Expression of Cellular Autophagy Factors [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 26-36.
[4]	DENG Xingmei, CAO Shuzhu, GUO Jia, ZHU Dexin, ZHAO Tianyi, CHAI Yingjin, ZHANG Wei, SHI Chao, JIA Sifeng, ZHANG Hui. Effect of Long Non-coding RNA Gm35082-202 on Cell Pyrodecay Induced by Brucella [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3599-3609.
[5]	XIAO Yangyang, MA Zhongchen, LI Ruirui, CHEN Chuangfu, ZHENG Wei, WANG Yong, WANG Pengyan. Bioinformatic Analysis and Polyclonal Antibody Preparation of Brucella Secreted Protein BspI [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3112-3121.
[6]	QIU Runhui, GUAN Feihu, WANG Zihang, GUO Jia, ZHU Dexin, ZHANG Wei, WEI Chunyan, HUO Mingkai, SUN Zhihua, ZHANG Hui. Construction and Biological Characteristics of Brucella Secreting Protein BMCO Gene Deletion Strain [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1630-1640.
[7]	JIANG Xiaoming, TANG Cheng, YUE Hua. Expression and Immunogenicity of P[1] Bovine Rotavirus A VP8 Recombinant Protein [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1970-1976.
[8]	QIU Runhui, GUAN Feihu, TAO Tingting, LI Jia, ZHAO Tianyi, DENG Xingmei, SHI Chao, SUN Zhihua, ZHANG Hui. Subcellular Localization and Screening of Host Interacting Proteins of Brucella Secreted Protein BPE005 [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1438-1448.
[9]	LIU Qingqing, WU Peng, LI Peidong, ZHANG Jiangwei, CHEN Chuangfu, XIAO Chencheng. Construction of Recombinant Adenovirus Expressing Porcine Reproductive and Respiratory Syndrome Virus N Protein and Its Immunogenicity Analysis [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 1024-1031.
[10]	WANG Yueli, SHAO Zhiran, YI Jihai, WANG Yong, WANG Zhen, CHEN Chuangfu. miR-145a-3p Regulates Brucella Induced Mouse Macrophage RAW264.7 Autophagy by Targeting ATG14 [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 1117-1125.
[11]	ZHANG Yanmin, ZHOU Yanan, CAO Lei, TIAN Yuan, LIU Xuping, TAN Wensong, ZHAO Liang. Expression and Immunogenicity Analysis of Classical Swine Fever Virus E2-GM-CSF Fusion Protein in HEK293T Cells [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 669-676.
[12]	ZHAO Jing, CHU Ying, LUO Jun, GUO Xiaofeng. Construction and Immunogenicity of Recombinant Rabies Virus with Canine Distemper Virus H Gene [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4776-4785.
[13]	XIE Shanshan, YANG Qin, DENG Xiaoyu, YI Jihai, WANG Zhen, CHEN Chuangfu. Effects of Mitochondrial Fusion Protein 2 Gene Interference and Overexpression on Macrophage Apoptosis Induced by Brucella [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 283-293.
[14]	YANG Jiangliu, QIAO Junwei, SONG Qingshan, JIA Fang. Prokaryotic Expression and Bioinformatics Analysis of Virulence Gene BvfA of Brucella S19 [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2746-2753.
[15]	WU Tongzhong, NUERSAILIKE Nusufu, HUANG Xin, HAN Mengli, ZHANG Xingxing, ZHANG Qian, WANG Xinhua, HE Yanhua, ZHONG Fagang. Construction of Recombinant Lactococcus Expressing E2 Protein of Bovine Viral Diarrhea Virus and Immunogenicity Analysis of the Protein [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2975-2981.