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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2021, Vol. 47 Issue (2): 251-260    DOI: 10.3785/j.issn.1008-9209.2020.05.190
动物科学与动物医学     
兔支气管败血波氏杆菌外膜囊泡的制备及其蛋白质成分分析
南黎1(),黄叶娥2,肖琛闻2,王志鹏2,韦强2,季权安2,李科2,刘燕2(),鲍国连2()
1.浙江师范大学化学与生命科学学院，浙江 金华 321000
2.浙江省农业科学院畜牧兽医研究所，杭州 310021
Preparation of outer membrane vesicles from rabbit Bordetella bronchiseptica and their protein composition analysis
Li NAN1(),Ye’e HUANG2,Chenwen XIAO2,Zhipeng WANG2,Qiang WEI2,Quan’an JI2,Ke LI2,Yan LIU2(),Guolian BAO2()
1.College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321000, Zhejiang, China
2.Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
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摘要：

本研究旨在确立兔波氏杆菌外膜囊泡（outer membrane vesicles, OMVs）最佳制备工艺。以兔波氏杆菌毒力较强菌株FX-1为研究对象，筛选出制备OMVs的最优方法，并对培养时间、抗生素添加量及过滤方法进行逐一优化。采用透射电镜（transmission electron microscope, TEM）、扫描电镜（scanning electron microscope, SEM）、纳米颗粒追踪分析（nanoparticle tracking analysis, NTA）等方法分析OMVs的理化性质；采用考马斯亮蓝法（Bradford法）、十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE）及液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry, LC-MS/MS）检测OMVs的蛋白质含量及组分。试验结果显示，超滤浓缩法是制备兔波氏杆菌OMVs的较适方法，采用该方法的最佳培养时间、头孢氨苄质量浓度和过滤方法分别为18 h、64 μg/mL和0.45 μm滤膜过滤一次。制得的OMVs呈纳米级球形囊泡，平均直径为127.83 nm±0.68 nm。蛋白质分析结果表明，OMVs上包含多种与兔波氏杆菌毒力和感染机制有关的蛋白质。该制备工艺能够显著提高兔波氏杆菌OMVs的产量，为其工业化生产提供了可能；对其结构和蛋白质成分的分析，为研发新型亚单位疫苗提供了科学依据。
关键词： 细菌外膜囊泡;  支气管败血波氏杆菌;  制备工艺;  蛋白质成分分析    
Abstract:

This study aimed to establish the optimal preparation method of outer membrane vesicles (OMVs) from rabbit Bordetella bronchiseptica (Bb). The FX-1 strain of rabbit Bb was used as the research object. Centrifugal ultrafiltration and ultrasonication were compared, and followed by the optimization of the preparation technics, such as culture time, antibiotic addition and filtering method. The physical and chemical properties of OMVs were examined with transmission electron microscope (TEM), scanning electron microscope (SEM) and nanoparticle tracking analysis (NTA), etc. The protein compositions were analysed with Bradford protein quantification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that centrifugal ultrafiltration was a better preparation method of OMVs, and the optimum culture time, cefalexin concentration and filtering method were 18 h, 64 μg/mL and filtration though 0.45 μm membrane once, respectively. The OMVs showed a spherical shape with the diameter of 127.83 nm±0.68 nm, containing a range of proteins associated with virulence and infection mechanisms. This preparation technique could significantly promote the production of OMVs, which making industrial production possible. Furthermore, analysis of the protein composition lays a foundation for novel subunit vaccine research and development.
Key words: bacterial outer membrane vesicles    Bordetella bronchiseptica    preparation technic    protein composition analysis
收稿日期: 2020-05-19 出版日期: 2021-04-25
CLC:  S 85  
基金资助: 国家兔产业技术体系岗位科学家项目(CARS-43-C-2);浙江省基础公益研究农业农村项目(LGN20C180003);浙江省农业科学院青年人才培养项目(2018R22R08E02)
通讯作者: 刘燕,鲍国连     E-mail: nanlio@163.com;35792191@qq.com;baoguolian@163.com
作者简介: 南黎（https://orcid.org/0000-0002-5809-2417），E-mail：nanlio@163.com
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南黎
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引用本文:

南黎,黄叶娥,肖琛闻,王志鹏,韦强,季权安,李科,刘燕,鲍国连. 兔支气管败血波氏杆菌外膜囊泡的制备及其蛋白质成分分析[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(2): 251-260.

Li NAN,Ye’e HUANG,Chenwen XIAO,Zhipeng WANG,Qiang WEI,Quan’an JI,Ke LI,Yan LIU,Guolian BAO. Preparation of outer membrane vesicles from rabbit Bordetella bronchiseptica and their protein composition analysis. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(2): 251-260.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2020.05.190    或    http://www.zjujournals.com/agr/CN/Y2021/V47/I2/251

图1  OMVs最优制备工艺的建立*表示在P＜0.05水平差异有统计学意义；**表示在P＜0.01水平差异有高度统计学意义。
图2  超滤浓缩法制OMVs的TEM、SEM和NTA观察结果A. OMVs的TEM观察结果；B. OMVs的SEM观察结果。
图3  SDS-PAGE分析超滤浓缩法和超声破碎法制得的OMVs蛋白条带1：分子标志物；2：超滤浓缩法；3：超声破碎法。

登录号

Accession No.

肽段质谱

匹配总数

Number of PSMs

蛋白质名称

Protein name

分子质量

Molecular mass/kDa

理论等电点

Calculated isoelectric point

A0A0H3LWR4746丝状血凝素/黏附素 Filamentous hemagglutinin/adhesin372.38.06
A0A0C6NZD9634黏附素 Adhesin328.58.59
A0A5A4M9X1602丝状血凝素 Filamentous hemagglutinin328.28.59
A0A5A4MAC4602丝状血凝素 Filamentous hemagglutinin381.48.54
A0A0H3LLW0599黏附素 Adhesin328.48.54
A0A4U9R3V8577丝状血凝素 Filamentous hemagglutinin328.58.46
A0A3T0NYS5438丝状血凝素 Filamentous hemagglutinin385.48.66
A0A5A4MEU4192

含β桶域的反向自主转运蛋白

Inverse autotransporter β-barrel domain-containing protein

164.55.74
A0A0R4J8N5188

假定外膜配体结合蛋白

Putative outer membrane ligand binding protein

164.65.71
A0A5A4M5C5137

含β桶域的自主转运外膜蛋白

Autotransporter outer membrane β-barrel domain-containing

protein

54.37.69
A0A5A4MM068660 kDa伴侣蛋白 60 kDa chaperonin57.45.24
A0A088B3B082鞭毛蛋白（片段） Flagellin (fragment)36.14.68
A0A0H3LML578鞭毛钩相关蛋白2 Flagellar hook-associated protein 247.75.00
A0A5A4M89069鞭毛蛋白（片段） Flagellin (fragment)40.24.75
A0A4U9RWQ966侵袭素 Invasin102.09.67
A0A4U9RYS560侵袭素 Invasin195.75.41
A0A0H3LIE045自主转运蛋白 Autotransporter237.59.39
A0A4U9RS6043绒毛蛋白 Fluffing protein228.39.39
A0A0H3LQ5936

N-乙酰胞壁-L-丙氨酸酰胺酶

N-acetylmuramoyl-L-alanine amidase

45.58.88
A0A3T0P40135Toll-Pal系统蛋白 Tol-Pal system protein47.19.47
A0A0C6PAD9、A0A4U9S6B533

DNA导向RNA聚合酶亚单位

DNA-directed RNA polymerase subunit

156.26.83
A0A3T0NU0233

含β桶域的自主转运外膜蛋白

Autotransporter outer membrane β-barrel domain-containing

protein

228.69.44
A0A0C6P9X8、A0A0H3LH4332

类枯草杆菌自主转运蛋白酶

Autotransporter subtilisin-like protease

99.49.63
A0A0H3LSC331二氢硫辛酸脱氢酶 Dihydrolipoyl dehydrogenase50.16.80
A0A0H3LU4930

2-氧戊二酸脱氢酶E1组分

2-oxoglutarate dehydrogenase E1 component

106.46.30
A0A0H3LXY6、A0A3T0P20628外膜蛋白A Outer membrane protein A21.08.73
A0A0C6P3B027溶血素激活蛋白 Hemolysin activator-like protein64.69.58
Q7WHP32430S核糖体蛋白S21 30S ribosomal protein S218.510.98
A0A0C6P01923周质硝酸盐还原酶 Periplasmic nitrate reductase92.98.12
A0A0H3LJ9422假定的TonB受体 Putative TonB-dependent receptor79.37.25
表1  超滤浓缩法制得的兔波氏杆菌OMVs主要蛋白质鉴定
图4  基于基因本体论（GO）的兔波氏杆菌OMVs蛋白的功能注释和富集分析A. 生物学过程；B. 分子功能；C. 细胞成分。
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