一种新的空肠弯曲菌外膜囊泡提取方法的建立

A New Method for Extracting Outer Membrane Vesicles of Campylobacter jejuni

空肠弯曲菌(Campylobacter jejuni)外膜囊泡(outer membrane vesicles,OMVs)是其在生长过程中分泌到细胞外的球状小泡,主要含有外膜蛋白及一些周质空间的物质,对细菌的生存、定植、细菌与宿主细胞间的交流及致病机制发挥重要的作用。因此,开发一种高效提取方法是研究空肠弯曲菌OMVs生物学功能的关键。该研究发现在微需氧条件下,使用MH培养基培养空肠弯曲菌15 h后提取OMVs最为合适。在最优条件下,采用超滤浓缩法从菌液中提取OMVs,并使用0.22μm的微孔滤头进一步过滤提取物除去鞭毛等杂质。为了表征所提取的OMVs质量,首先使用透射电镜技术,发现所提取的OMVs具有典型形态,大小在50~300 nm之间,所含杂质较少。SDS-PAGE电泳结果表明OMVs内部含有大量蛋白,含量可达40.50 mg/mL。最后利用外膜蛋白抗体成功证明所提取的物质的主要成分为OMVs。该研究成功建立了一种从液体培养基中提取空肠弯曲菌OMVs的提取体系,多种分析方法证明其提取效率高、质量较好,适用于后续空肠弯曲菌OMVs生物功能相关研究。

Outer membrane vesicles(OMVs)of Campylobacter jejuni are globular vesicles secreted to the outside of cellsduring the growth process,whichmainly contain outer membrane proteins and some periplasmic substances,and play an important role in the survival,colonization,communication between bacteria and host cells,and pathogenic mechanisms.Therefore,the development of an efficient extraction method is the key to studying the biological function of OMVs.This study found that it is most appropriate to extract OMVs after culturing C.jejuni with MH medium for 15 h under microaerobic conditions.Under optimal conditions,the ultrafiltration concentration method was used to extract OMVs from the bacterial liquid,and the extract was further filtered using a 0.22μm microporous filter to remove flagella and other impurities.In order to characterize the quality of the extracted OMVs,a transmission electron microscope was used.It was found that the extracted OMVs had a typical morphology(with their size ranging from 50 to 300 nm),and contained less impurities.The results of SDS-PAGE showed that OMVs contained a large amount of proteins(whose total content was 40.50 mg/mL).Finally,the outer membrane protein antibody was used to prove successfully that the main components of the extracted material were OMVs.In this study,an extraction system for extracting C.jejuni OMVs from liquid culture medium was successfully established.Various analytical methods have proved that its extraction efficiency is high and its quality is good.It is suitable for subsequent studies on the biological functions of C.jejuni OMVs.