Characterization of outer membrane vesicles from Escherichia coli BZB1107 and BL21-DE3 strains

Abstract

Vaccine is a prerequisite enhancer of acquired immunity used to protect against pathogen. The outer membrane vesicles (OMVs) are currently used for developing vaccine delivery system. Nevertheless, OMVs also contains outer membrane proteins (OMPs), which may be able to alter immunogenic effects and stability. The main objective of this study was to characterize OMVs for vaccine delivery system. Here, we compared OMV produced from BL21-DE3 and BZB1107 strains of Escherichia coli with or without expression of outer membrane proteins (OMPs), respectively. ClyA-GPF-6xhistidine tag gene was inserted into the vector. GFP was used as a representative antigen and an outer membrane protein, cytolysin A (ClyA)whereas 6xhistidine tag was used for detection. Sanger sequencing of engineered plasmid revealed a correct sequence of plasmids encodingClyA-GFP-6xHistidine tag. After the production of OMVs containing ClyA-GFP, dynamic light scattering (DLS) revealed the size distribution of OMVs from BZB1107 and BL21-DE3 were 40 to 200 nm and 43 to 300 nm in diameter, respectively. In addition, SDS-PAGE and western blot analysis were able to detect band of ClyA-GFP-6xHistidine tag at 62 kDa in both types of OMVs. Transmission electron microscope revealed the morphology and size of both OMVs, indicating that size of OMVs from BZB1107 were smaller than those from BL21DE3. ELISA assay indicated that ClyA-GPF-6xhistidine tag localized on the surface of both types of OMVs. Stability assay showed that OMVs isolated from BZB1107 and BL21-DE3 were resistant to temperature-challenged conditions, but only OMVs from BZB1107 was not affected by EDTA-induced destabilization, suggesting that OMPs may be involved in maintaining OMV stability. Although there are some differences and a new OMV platform need to more characterized, it is possible that OMV from BZB1107(without OMP expression) may represent a new platform of OMV without immunogenic hyper-reactivity. In addition, stability of OMVs from BZB1107 was stable in response to various temperature exposure in which this property may be further developed to be an efficient vaccine in the future.