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The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.
Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane vesicles (OMVs) are mainly secreted by gram-negative bacteria during average growth, with a nano-sized diameter and self-adjuvant activity, which may be ideal for vaccine delivery. OMVs have functioned as a multifaceted delivery system for proteins, nucleic acids, and small molecules. To take full advantage of the biological characteristics of OMVs, bioengineered Escherichia coli-derivedĀ OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a "Plug-and-Display" vaccine platform. The SpyCatcher (SC) and SpyTag (ST) domains in Streptococcus pyogenes were applied to conjugate OMVs and RBD. The Cytolysin A (ClyA) gene was translated with the SC gene as a fusion protein after plasmid transfection, leaving a reactive site on the surface of the OMVs. After mixing RBD-ST in a conventional buffer system overnight, covalent binding was formed between the OMVs and RBD. Thus, a multivalent-displaying OMV vaccine was achieved. By replacing with diverse antigens, the OMVs vaccine platform can efficiently display a variety of heterogeneous antigens, thereby potentially rapidly preventing infectious disease epidemics. This protocol describes a precise method for constructing the OMV vaccine platform, including production, purification, bioconjugation, and characterization.
As a potential vaccine platform, outer membrane vesicles (OMVs) have attracted more and more attention in recent years1,2. OMVs, mainly secreted naturally by gram-negative bacteria3, are spherical nanoscale particles composed of a lipid bilayer, usually in the size of 20-300 nm4. OMVs contain various parental bacterial components, including bacterial antigens and pathogen-associated molecular patterns (PAMPs), which serve as solid immune potentiators5. Benefiting from their unique components, natural vesicle structure, and great genetic en....
1. Plasmid construction
The flowchart for this protocol is shown in Figure 1. This protocol could be a general approach to utilizing OMVs as a vaccine platform; one only needs to choose the appropriate expression systems based on the type of antigens.
Figure 2Ā provides a feasible plasmid design scheme. The SC gene is connected with the ClyA gene via a flexible linker, while ST connects to the 5' terminal of the RBD gene with a His-tag gene for purif.......
To create a "Plug-and-Display" nanoparticle vaccine platform, SC-fused ClyA was expressed in BL21(DE3) strains, which is one of the most widely used models for recombinant protein production because of its advantages in protein expression24, so that there would be enough SC fragment displaying on the surface of the OMVs during the process of bacteria proliferation. At the same time, an ST-fused target antigen was prepared for the chemical coupling between the antigens and OMVs. This experi.......
This work was supported by the Key Program of Chongqing Natural Science Foundation (No. cstc2020jcyj-zdxmX0027) and the Chinese National Natural Science Foundation Project (No. 31670936, 82041045).
....Name | Company | Catalog Number | Comments |
Ampicillin sodium | Sangon Biotech | A610028 | |
Automated cell counter | Countstar | BioTech | |
BCA protein quantification Kit | cwbio | cw0014s | |
ChemiDoc Touching Imaging System | Bio-rad | ||
Danamic Light Scattering | Malvern | Zetasizer Nano S90 | |
Electrophoresis apparatus | Cavoy | Power BV | |
EZ-Buffers H 10X TBST Buffer | Sangon Biotech | C520009 | |
Goat pAb to mouse IgG1 | Abcam | ab97240 | |
High speed freezing centrifuge | Bioridge | H2500R | |
His-Tag mouse mAb | Cell signaling technology | 2366s | |
Imidazole | Sangon Biotech | A600277 | |
Isopropyl beta-D-thiogalactopyranoside | Sangon Biotech | A600118 | |
Ni-NTA His-Bind Superflow | Qiagen | 70691 | |
Non-fat powdered milk | Sangon Biotech | A600669 | |
OPM-293 cell culture medium | Opm biosciences | 81075-001 | |
pcDNA3.1 RBD-ST plasmid | Wuhan genecreat biological techenology | ||
Phosphate buffer saline | ZSGB-bio | ZLI-9061 | |
Polyethylenimine Linear | Polysciences | 23966-1 | |
Prestained protein ladder | Thermo | 26616 | |
pThioHisA ClyA-SC plasmid | Wuhan genecreat biological techenology | ||
PVDF Western Blotting Membranes | Roche | 03010040001 | |
Quixstand benchtop systems (100 kD hollow fiber column) | GE healthcare | ||
SDS-PAGE loading buffer (5x) | Beyotime | P0015 | |
Sodium chloride | Sangon Biotech | A100241 | |
Supersignal west pico PLUS (enhanced chemiluminescence solution) | Thermo | 34577 | |
Suspension instrument | Life Technology | Hula Mixer | |
Transmission Electron Microscope | Hitachi | HT7800 | |
Tryptone | Oxoid | LP0042B | |
Ultracentrifuge | Beckman coulter | XPN-100 | |
Ultraviolet spectrophotometer | Hitachi | U-3900 | |
Yeast extract | Sangon Biotech | A610961 |
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