Sharif Elham, Eftekhari Zohre, Mohit Elham
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Student Research Committee, Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Curr Microbiol. 2021 Apr;78(4):1602-1614. doi: 10.1007/s00284-021-02414-y. Epub 2021 Mar 9.
Outer membrane vesicles (OMVs) are nanosized spherical blebs derived from the outer membrane of gram-negative bacteria. Outer membrane vesicles (OMVs) play important roles in various physiological functions of bacteria. They can be applied as native vaccines or vaccine adjuvants. The objective of this study was to determine the appropriate growth phase and isolation method for OMV separation from ClearColi™, an endotoxin-free strain of E. coli. It was demonstrated that the yield of OMVs is increased while the bacteria are growing. Herein, although total protein concentration of OMVs isolated from the stationary phase is more than other phases; the pre-stationary phase was selected for OMV isolation due to release of smaller size of OMVs as compared to other phases. In the current study, to obtain OMVs with high yield, proper size, and homogeneity, different concentration methods including protein precipitation by ammonium sulfate (AS) and ultrafiltration (UF) were combined to ultracentrifugation (UC) or precipitation-based exosome isolation kit. Among the examined isolation methods, AS (70%) + UC resulted in the highest yield of OMVs. The TEM results demonstrated bilayer round-shaped OMVs isolated by this method. Although AS (70%) + kit resulted in more heterogeneous in size and larger OMVs as compared to AS (70%) + UC, it is applicable when high yield of OMVs is required and UC is not available. Totally, isolation of ClearColi™ OMVs from pre-stationary phase using AS (70%) + UC with enhanced yield can be applied in vaccine research studies.
外膜囊泡（OMVs）是源自革兰氏阴性菌外膜的纳米级球形泡。外膜囊泡在细菌的各种生理功能中发挥着重要作用。它们可用作天然疫苗或疫苗佐剂。本研究的目的是确定从无内毒素的大肠杆菌菌株ClearColi™中分离OMV的合适生长阶段和分离方法。结果表明，在细菌生长过程中，OMV的产量会增加。在此，虽然从稳定期分离的OMV的总蛋白浓度高于其他阶段；但由于与其他阶段相比，稳定前期释放的OMV尺寸较小，因此选择稳定前期进行OMV分离。在本研究中，为了获得高产率、合适尺寸和均一性的OMV，将不同的浓缩方法（包括硫酸铵（AS）沉淀蛋白和超滤（UF））与超速离心（UC）或基于沉淀的外泌体分离试剂盒相结合。在所研究的分离方法中，AS（70%）+UC产生的OMV产量最高。透射电镜结果显示通过该方法分离出双层圆形OMV。虽然与AS（70%）+UC相比，AS（70%）+试剂盒产生的OMV尺寸更不均一且更大，但在需要高产率的OMV且无法进行UC时，它是适用的。总的来说，使用AS（70%）+UC从稳定前期分离ClearColi™ OMV并提高产量，可应用于疫苗研究。
