Sars cov 2 spike rbd

5 × 10⁵ HEK293FT cells/well were plated on a 6 well plate (Corning) and were transiently transfected with 2 µg GLB-COV2-043 mRNA encoding SARS-CoV-2 full length wild-type (Wuhan-Hu-1) Spike protein using Lipofectamine MessengerMax (Thermo Fisher) following the supplier recommendations. 20 h post transfection, the cells were lysed using RIPA lysis buffer (Thermo Fisher) with protease inhibitors (Thermo Fisher) and DNAse I (NEB). The cell lysates were collected after centrifugation (4 °C, 12,000 g for 15 min), reduced with DTT (Thermo Fisher) and denatured in sample buffer (Thermo Fisher) upon heating at 95 °C for 5 min. Cell extracts were run on a 4–12% Bis–Tris gel (Invitrogen) and transferred to a nitrocellulose membrane (Bio-Rad) using Trans-Blot Turbo transfer system (Bio-Rad). The membrane was then blocked with Intercept blocking buffer (Licor) for 1 h at room temperature and incubated with SARS-CoV-2 Spike RBD or anti-S2 antibodies (Sino Biological) at 4 °C overnight. The following day, the membrane was washed with 1 × PBS-0.2%Tween-20 (Fisher) and incubated with a HRP labeled goat anti-rabbit secondary antibody (Abcam) at room temperature for 1 h. The membrane was washed again with PBS-0.2% Tween-20 buffer, and the signal was detected using an Odyssey CLx Infrared Imaging System.

An Octet system (Octet RED96, ForteBio, United States) was employed for biolayer interferometry (BLI) to determine the binding kinetics and affinity of compounds or extracts with the proteins of SARS-CoV-2 spike RBD and human ACE2 (Sino Biological, Beijing, China). Either the recombinant His-tag human ACE2 (25 μg/mL aqueous solution) or recombinant His-tag SARS-CoV-2 spike RBD (25 μg/mL aqueous solution) was immobilized onto the surface of nitrilotriacetic acid (Ni-NTA) biosensors (Fortebio, United States) that were dipped in a range of concentrations of compounds or extracts. Background signal was subtracted from all samples by using a reference biosensor loaded with either SARS-CoV-2 spike RBD or ACE2, which did not receive compounds or extracts under identical condition. The subtracted sensorgrams were then fitted to a 1:1 binding model to calculate the resulting equilibrium dissociation constant (K_D) value for this interaction. All experiments were repeated at least three times and K_D values were presented with mean ± SD.

Cells were washed with PBS and lysed by RIPA buffer (Thermo Scientific, 89901) supplemented with 100× protease inhibitor cocktail and phosphatase inhibitor (Thermo Scientific, 78420), followed by a 10-min incubation on ice. Cell lysates were then subjected to centrifugation at 13,500 RPM for 10 min at 4°C to remove cell debris and chromatids. The protein samples were then boiled in 2× Laemmli Sample Buffer (Bio-Rad, #1610737EDU) containing 5% β-mercaptoethanol at 95°C for 5 min. Prepared samples were run in 4%–12% gels and transferred onto nitrocellulose membranes. Membranes were blocked in 5% BSA in TBS +0.1% Tween-20 (TBST) at room temperature before incubation at 4°C overnight with primary antibodies: SARS-CoV-2 spike RBD (Sino Biological, 40592-T62), GAPDH (BioLegend, 631402), calpain-2 (Cell Signaling Technology, 2539), ACE2 (R&D Systems, MAB933), S2 (Sino Biological, 40590-T62), and V5 (Cell Signaling Technology, 13202S). Membranes were then washed three times with TBST and incubated in secondary antibodies accordingly: anti-mouse HRP-linked IgG (Cell Signaling Technology, 7076S) or anti-rabbit HRP-linked IgG (Invitrogen, A27036) diluted in 5% BSA in TBST at room temperature for 1 h. After the secondary antibody incubation, the membranes were washed three times with TBST and visualized by using Chemi-Doc imaging system (Bio-Rad).