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
1. Plasmid construction Insert DNA encoding SpyCatcher sequence (Figure 1) into an ampicillin-resistant pThioHisA-Cytolysin A (ClyA) plasmid (see Table of Materials) between the BamH I and Sal I sites to construct the plasmid pThioHisA ClyA-SC following a previously published report. Ligate the synthesized SpyTag-SARS-CoV-2 receptor-binding domain (RBD)-polyhistidine tag (Histag) fusion gene (Figure 1) into a pcDNA3.1 plasm…
Representative Results
Figure 1: Plasmid sequences used in the present study.
Offenlegungen
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)