To generate the secreted S-trimer fusion proteins of the prototype and BA.1 variant carrying 453F and 501 T mutations, these two segments of the spike protein ectodomain (amino acid residues 16–1209 with a 6P mutation) were separately fused with an N-terminal signal peptide and a C-terminal region containing the T4 foldon, human rhinovirus (HRV) 3C, and Twin-strep-His10. These sequences were codon-optimized and synthesized at Tsingke Biotechnology before being individually inserted into the pCMV-GS expression vector. The plasmids were transfected into CHO-K1 cells.
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