FIG.1.

Antigenic analysis of the S protein. (A) Potential antigenic peptides predicted by EMBOSS:Antigenic. The scores are related to the probability that the amino acid sequence is an antigenic determinant on the basis of empirical data and the distribution of amino acids (9). (B) BLAST analysis of the S protein sequences of SARS CoV and HCoV 229E was performed with the full-length amino acid sequences of both proteins at the National Center for Biotechnology Information website. The letters between the two sequences are conserved residues, and the plus signs indicate homologous changes. The starting point corresponds to the first amino acid aligned by BLAST, and only the beginning portion of the alignment is shown here. The shaded region showed significant homology (60%) in the receptor binding region of the HCoV 229E S protein. (C) The codons for S-II were optimized for usage in E. coli, and 18 oligonucleotides grouped with sticky overlap ends were synthesized and annealed together as nicked double-stranded DNA and ligase treated. The synthesized cDNA was cloned into the expression vector pET100/D-TOPO with a His₆ tag and an EK recognition site at the N terminus (Invitrogen), and protein expression was induced with IPTG in BL21/ED3 host cells. The inclusion bodies containing S-II were dissolved in 8 M urea, refolded, and purified by Ni affinity chromatography. The yield of purified, refolded, soluble S-II was >2 mg/liter. Lanes: 1, molecular mass marker; 2, denatured S-II expressed in E. coli; 3, purified, refolded S-II. (D) The purified, refolded S-II was biotinylated and used as a probe for specific binding to the surface of Vero cells. Vero E6 cells (10⁶; American Type Culture Collection) were first incubated with a 20-μl volume of 10 μg of biotinylated S-II or a control His₆-tagged S.Tag protein (Novagen) per ml at RT for 30 min and then further incubated with 20 μl of PE-conjugated streptavidin. The binding of S-II to Vero cells was demonstrated by flow cytometry analysis. S-II, solid; control, open. S-II showed selective binding to Vero cells compared to two recombinant protein fragments covering aa 145 to 480 (S-I) or 601 to 973 (S-III). S-RBD, S protein receptor binding domain. (E) To demonstrate dose-dependent binding of S-II to Vero cells, Vero cells were incubated with the indicated concentration of the S-II fragment (solid) or a control (open) and MFI was determined. (F) The same flow cytometry analysis as in panel D, using fragments S-I and S-III.