To uncover the amino acids important for ACE2 receptor recognition, ACE2 ecotodomain (residues Q18 to S740) gene, with an N-terminal IL-10 signal peptide, tagged with human immunoglobulin G1Fc (IgG1Fc) and His tag at the C terminus, was cloned into the pcDNA 3.4 vector. Codon-optimized RBD (residues V320 to G550) gene fragment, with an N-terminal IL-10 signal peptide, tagged with His tag at the C terminus, was cloned into the pcDNA 3.4 vector. Three SARS-CoV-2 RBD mutants were constructed. For mutant RBD-(core), amino acids R319 to N437 of core region in the SARS-CoV-2 RBD were substituted by the corresponding region of SARS-CoV strain Tor2 (GenBank ID: AAP41037.1). For mutants RBD-(RBM-R2) and RBD-(RBM-R3), residues L452 to K462, and residues T470 to T478 of the RBM region in the SARS-CoV-2 RBD were mutated into the corresponding regions of SARS-CoV strain Tor2, respectively. For single-point mutations of RBD (Q498A), RBD (V503A), and RBD (Y505A), RBD residues Q498, V503, and Y505 were substituted by Ala, respectively. All mutant plasmids were constructed using the MutExpress II Fast Mutagenesis Kit V2 (Vazyme, China) according to the manufacturer’s instruction. The proteins were generated using HEK293F expression system and purified as described above.

Anti-RBD polyclonal antibody and monoclonal antibody (MAb) 1A10 were prepared by immunizing BALB/c mice with recombinant SARS-CoV-2 RBD fused with a C-terminal mouse IgGFc tag (Sino Biological Inc., Beijing, China) using previously described protocols (63).

The purified RBD mutants were tested by ELISA for reactivity with the receptor ACE2. Briefly, ELISA plates were coated with 100 ng per well of the purified RBD mutants in PBS at 37°C for 2 hours and then blocked with 5% milk in PBS–Tween 20. Next, the plates were incubated with 50 ng per well of ACE2-hFc fusion protein, 50 μl per well of culture supernatant of hybridoma 1A10, or 50 μl per well of mouse anti-RBD sera (diluted at 1/1000) at 37°C for 2 hours. After washing, the corresponding secondary antibodies, horseradish peroxidase (HRP)–conjugated anti-human IgG1 (Abcam, USA) or HRP-conjugated anti-mouse IgG (Sigma-Aldrich, USA), were added and incubated at 37°C for 1 hour. After washing and color development, absorbance at 450 nm was determined.

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