Preparation and Identification of SARS-CoV-2 VLPs

The preparation and identification of SARS-CoV-2 VLPs are described previously (Mi et al., 2021 (link)). In brief, the codon-optimized S, E, and M genes of SARS-CoV-2 (GenBank accession no. MN908947.3) were cloned into the pFastBac triple expression vector. Recombinant baculovirus was produced using the Bac-to-Bac system (ThermoFisher Scientific, USA). VLPs were then obtained by infecting ExpiSf9™ insect cells with the recombinant baculovirus and purified via density gradient centrifugation.
SARS-CoV-2 VLPs were adsorbed onto a 400-mesh carbon-coated film for 2 min, which was then stained with 1% phosphotungstic acid for 60 s. After staining, VLP morphology was visualized using FEI Talos F200C transmission electron microscope (FEI, Czech Republic). For immunoelectron microscopy, VLPs were captured on carbon-coated copper grids. The grids were incubated with rabbit anti-spike polyclonal antibody (1:50 dilution; SinoBiological, China) at room temperature for 1 h, followed by treatment with goat anti-rabbit immunoglobulin G (1:20 dilution) (whole)-gold conjugate (10 nm) (BOSTER, China). Finally, the negative staining of the grids was performed using 1% phosphotungstic acid. VLPs were observed on the transmission electron microscope (FEI, Czech Republic) at 200 kV and 100–200 kfold magnification.