Construction, expression, and purification of recombinant protein
The construction, expression, and purification of recombinant RBD proteins of SARS-CoV-2, SARS-CoV, and MERS-CoV were performed as previously described with some modifications.27,28 Briefly, genes encoding residues 331-524 of SARS-CoV-2 S protein, residues 318-510 of SARS-CoV S protein, or residues 377-588 of MERS-CoV S proteins, were amplified by PCR using codon-optimized SARS-CoV-2 S protein (GenBank accession number: QHR63250.1), SARS-CoV S protein (GenBank accession number: AY278488.2), or MERS-CoV S protein (GenBank accession number: AFS88936.1), as respective template, and fused into pFUSE-hIgG1-Fc2 expression vector (hereinafter named hFc; InvivoGen, San Diego, CA). The RBD proteins were expressed in human embryonic kidney (HEK)293T cells, secreted into cell culture supernatants, and purified by protein A affinity chromatography (GE Healthcare, Marlborough, MA).