Construction and characterization of spike expression plasmids derived from non-SARS HCoVs. (A) Construction scheme of spike (S) fragment expression plasmids derived from four individual HCoVs (−229E, -OC43, -NL63, and -HKU1). (B) Characterisation of S proteins of individual HCoV expression plasmids by western blot using anti-S mouse serum from DNA vaccination. Forty-eight hours after 293 T cell transfection with the above individual HCoV expression plasmids, the cell lysates were collected for SDS-PAGE and western blot. (C) Characterisation of S protein by indirect immune fluorescence assay (IFA) using anti-S serum from mice immunised with individual HCoV expression plasmids. The control was transfected with pVRC-8303 plasmid and detected with mixed anti-S mouse serum of a non-SARS CoV.