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

Preparation of outer membrane vesicles from rabbit Bordetella bronchiseptica and their protein composition analysis

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、64μg/mL和0.45μm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。

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,64μg/mL and filtration though 0.45μm 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.