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

摘要/Abstract

摘要： 【目的】探索布鲁氏菌外膜囊泡（OMVs）在亚单位疫苗上的应用前景，制备布鲁氏菌OMVs亚单位疫苗，评估布鲁氏菌OMVs的免疫原性。【方法】利用高速离心法制备羊种布鲁氏菌OMVs，通过透射电镜、SDS-PAGE和生物信息学分析对制备的OMVs进行形态和组分分析；使用纳米佐剂（LDH）和弗氏佐剂乳化OMVs后免疫小鼠，通过间接ELISA方法检测免疫后7、14、21、28和35 d小鼠血清中的特异性抗体，利用小鼠IgG ELISA检测试剂盒检测免疫后小鼠血清中IgG抗体水平，评估OMVs诱导产生的体液免疫水平；通过小鼠脾脏淋巴细胞分离与细胞因子白细胞介素4（IL-4）和γ-干扰素（IFN-γ）测定及小鼠外周血T淋巴细胞亚群的流式细胞术测定评估OMVs免疫小鼠的细胞免疫水平。【结果】成功提取并纯化了布鲁氏菌OMVs，其直径大小为20~160 nm。生物信息学分析结果显示，OMVs主要组成蛋白OMP25、OMP31、OMP16、OMP19、BP26及SOD均属于亲水性蛋白和抗原性蛋白，且存在多个B细胞和T细胞优势抗原表位。与PBS组相比，OMVs免疫小鼠后，不同佐剂组均可诱导小鼠产生高水平的特异性抗体和IgG抗体（P<0.01）；同时OMVs可显著诱导小鼠脾脏淋巴细胞产生IFN-γ（P<0.05），并促进了CD4⁺ T细胞的表达（P<0.05）。【结论】本研究证实了羊种布鲁氏菌OMVs具有较强的免疫原性，能诱导小鼠产生强烈的体液免疫和细胞免疫反应，为布鲁氏菌新型亚单位疫苗的开发奠定了理论基础。

关键词: 布鲁氏菌; 外膜囊泡(OMVs); 免疫原性

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

中图分类号:

S852.61^＋4

徐朕宇, 徐锦凤, 邓肖玉, 王月丽, 何金科, 谢珊珊, 王震, 易继海, 王勇, 苗玉和, 崔丽瑾, 陈创夫. 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 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.

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