Anti sars cov 2 s1

The cell was washed twice with PBS and lysed using RIPA (Beyotime) containing 1% cocktail. Consequently, whole-cell lysates were obtained via ice lysis for 30 min. A total of 10% SDS loading buffer was added. Afterward, the lysates were electrophorized in polyacrylamide gel and transferred to the nitrocellulose membrane. The membrane was blocked with 5% BSA for 1 h and subsequently incubated with primary antibody for 3 h. The primary antibodies used were indicated as follows: anti-MDA5 (Proteintech, 21775-1-AP), anti-RIG-I (ABclonal, A18003), anti-LGP2 (ABclonal, A8257), anti-GAPDH (Proteintech, 60004-1-lg), anti-SARS-CoV-2 S1 (Sino Biological, 40591-T62), SARS-CoV-2 N (Sino Biological, 40143-MM05), anti-Flag (Cell Signaling Technology, #14793), and anti-hACE2 (Sino Biological, 10108-RP01). Following three washes with TBST, the membranes were incubated with the appropriate secondary antibody. The membranes were finally visualized using the ChemiDoc MP (Bio-Rad).

To determine the presence of RBD on the viral surface of rLS1-HN-RBD, and the presence of the S1 subunit on rLS1-S1-F viruses, virion particles were purified with a 25% sucrose cushion. Vero-E6 cells were harvested and washed with DPBS with 5% FBS. Approximately, 1 × 10⁶ cells were blocked with DPBS with 5% of naïve mouse serum for 30 min at 37 °C. Then, the cells were incubated with rLS1 (0.36 mg/mL), rLS1-S1-F (0.09 mg/mL) or rLS1-HN-RBD (0.2 mg/mL) purified viruses for 30 min at 37 °C. To remove the residual viral particles not attached to Vero-E6, the cells were washed with DPBS and 5% FBS twice. Subsequently, cells were incubated with rabbit monoclonal antibody anti-SARS- CoV-2 S1 (1:200) (Sino Biological, Beijing, China) as primary antibody for 1h at 37 °C, followed by goat anti-rabbit IgG Alexa Fluor® 488 (1:200) (Abcam, Cambridge, MA, USA) as secondary antibody.
Finally, the cells were analyzed in FACS Canto II (BD Biosciences, USA) flow cytometer. The data obtained were analyzed using the software FlowJo v.10.6 (BD Biosciences, USA), where the percentage of positive cells was taken to indicate detection of the SARS-CoV-2 S1 subunit or RBD on the viral surface of viruses bound to Vero-E6.