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Primer pairs were N gene: NF, 5'-GCCGAATTCATGTCTGATAATGGACCCCA-3' and NR, 5'-GCGCGT CGACGTTATGCTGAGT TGAATCA-3'; SIa gene (aa residues 1-460 of the S protein): S1F, 5’-GCGGAATTCATGTTTATTTTCTTATTATTTCTTAC-3’ and S1R, 5’-GCG CTCGAGGAAAGGCACATT AGATATGTCT-3’; SIb gene (aa residues 416-846 of the S protein): S2F, 5’-GCGGAATTCATGGGT TGTGTCCTTGCTTGGA-3’ and S2R, 5’-GCGCTCGAGCAGAGGTGGCAACACTGTA AGT-3’; SII gene (aa residues 815-1255 of the S protein): S3F, 5’-GCGGAATTCATGAAG CAATATGGCGAATGC-3’ and S3R, 5’-GCGC TCGAGTGTGTAATGTAATT TGACACCC-3’. PCR products were subcloned into pGEX-4T-1 (Amersham Biosciences, NJ) or pET-29a vectors (Norvogen). Recombinant proteins (RP) we r e i n d u c e d i n t o B L 2 1 c e l l s w i t h i s o p r o p y l - ²- D - t h i o g a l a c t o p y r a n o s i d e ( I P T G ) ( S i g m a - A l d r i c h ) . F o r i n d u c t i o n , t h e o p t i c d e n s i t i e s o f B L 2 1 c e l l s c o n t a i n i n g t h e p G E X - N P , p G E X - S I a , p G E X - S i b o r p G E X - S I I p l a s m i d s w e r e 0 . 6 - 0 . 7 . F o l l o w i n g I P T G i n d u c t i o n a t 3 7 ° C f o r 2.5 h, bacterial cells were collected by centrifugation at 6,000 rpm at 4°C for 15 min. Cell pellets were washed twice with ice-cold phosphate buffered saline (PBS pH 7.4) (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), resuspended in 20 ml PBS, French pressed, centrifuged at 16,000 x g for 30 min at 4ºC, and resuspended in 5 ml 8M urea. Resulting supernatant was applied to gels using a SDS-polyacrylamide gel process. Following overnight dialysis against PBS, proteins were concentrated with a Centricon Plus-10 concentrator (Millipore, MA, USA). RP concentrations were measured with a Pierce bicinchoninic acid protein assay reagent (Pierce, IL); purity was analyzed using samples with a 12.5% sodium dodecyl sulfate-polyacrylamide mini-gel (Bio-Rad Laboratories, CA). Figure legends: Figure 1. Construction of plasmids containing specific regions of the SARS-CoV spike (S) gene. We cloned the S gene in a pGEX system for prokaryotic expression. Gene fragments were generated by one-tube RT-PCR. For cloning to the pGEX system, each PCR product was gel-purified, digested with EcoRI and XhoI, and ligated into a pGEX-4T-1 vector. Each construct was sequenced using a dye terminator cycle sequencing core kit on a DNA sequencer. Figure 2. Construction of plasmids containing specific regions of the SARS-CoV nucleocapsid (N) gene. We cloned the N gene in a pGEX system for prokaryotic expression. Gene fragments were generated by one-tube RT-PCR. For cloning to the pGEX system, each PCR product was gel-purified, digested with EcoRI and XhoI, and ligated into a pGEX-4T-1 vector. Each construct was sequenced using a dye terminator cycle sequencing core kit on a DNA sequencer. Figure 3. Antibody reactivity to SARS-CoV spike (S) and nucleocapsid (N) proteins using immunoblotting assays. Shown are results from immunoblotting assays of S and N recombinant proteins (arrows) detected by anti-S or anti-N mouse anti-sera (lane 2), SARS-positive sera (lane 1), or mouse control sera (lane 3). Figure 4. FACS analyses of ACE2 and Fc Receptors. Raji and THP-1 cells were stained with mouse monoclonal antibodies against human CD16, CD32, CD64 and isotype controls. Cells were washed and incubated for 30 min at 4°C with FITC-conjugated goat anti-mouse antibodies. Stained cells were analyzed by LSRII flow cytometry. Results shown are from one representative experiment out of three performed. 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