Generating a Multivalent-Displaying Outer Membrane Vesicle Vaccine
Abstract
Source: Feng, R., et al. A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain. J. Vis. Exp. (2022).
This video demonstrates a method to generate multivalent-displaying outer membrane vesicle (OMV) vaccines. Mammalian cells are transfected to express and secrete viral antigens fused to a SpyTag and a polyhistidine tag. These antigens are then purified using a nickel-nitrilotriacetic acid (Ni-NTA) affinity column. The purified antigen is mixed with outer membrane vesicles expressing surface-bound SpyCatcher (SC) protein. Multiple antigens covalently attach to SpyCatchers via their SpyTags (ST), creating OMVs that display the antigen, serving as a multivalent vaccine.
Protocol
Representative Results
Disclosures
The authors have nothing to disclose.
Materials
| Ampicillin sodium | Sangon Biotech | A610028 | |
| Automated cell counter | Countstar | BioTech | |
| BCA protein quantification Kit | cwbio | cw0014s | |
| Electrophoresis apparatus | Cavoy | Power BV | |
| 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 | |
| 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 | ||
| Quixstand benchtop systems (100 kD hollow fiber column) | GE healthcare | ||
| SDS-PAGE loading buffer (5x) | Beyotime | P0015 | |
| Sodium chloride | Sangon Biotech | A100241 | |
| Suspension instrument | Life Technology | Hula Mixer | |
| Tryptone | Oxoid | LP0042B | |
| Ultracentrifuge | Beckman coulter | XPN-100 | |
| Ultraviolet spectrophotometer | Hitachi | U-3900 | |
| Yeast extract | Sangon Biotech | A610961 |
Tags
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