Immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus

Abstract

Severe acute respiratory syndrome (SARS) is a novel infectious disease caused by the SARS-associated coronavirus (SARS-CoV). There are four major structural proteins in the SARS-CoV, including the nucleocapsid, spike, membrane, and small envelope proteins. In this study, two sets of truncated fragments of spike protein were generated, the first were approximately 210-bp nonoverlapping fragments and the second were overlapping segments of 750 to 900 bp. From these 23 fragments, we identified a fragment of 259 amino acids (amino acids 441 to 700) that is a major immunodominant epitope. This fragment was highly expressed, and the purified fragment C could detect all 33 SARS patient serum samples tested, collected from 7 to 60 days after the onset of fever, but had no reactivity with all 66 healthy human serum samples tested. Thus, fragment C of spike protein was identified as an immunodominant antigen and could be used for serological detection of SARS-CoV infection.

FIG. 1.

SDS-PAGE and Western blot analysis of 18 nonoverlapping fragments covering the whole S protein expressed as GST fusion proteins. (A) Schematic diagram of 18 nonoverlapping fragments within the S gene. (B) SDS-PAGE of the 18 induced GST fusion fragments. GST fusion proteins around 32 kDa were expressed in all of the induced cells. M, protein marker; BL21, control BL21 cellular extract; S1 to S18, total cellular extracts harvested 4 h post-IPTG induction of the BL21 cells transformed with recombinant pGEX constructs bearing s1 to s18, respectively. (C) Western blot analysis of the expressed GST fusion fragments with monoclonal mouse anti-GST antibody. M, protein marker; GST, control GST protein; N, control BL21 cellular extract; S1 to S18, samples as described for panel A. The arrows indicate the expressed fusion proteins.

FIG. 2.

Expression of five overlapping domains within the S protein in SF9 cells. (A) Schematic diagram of five overlapping fragments within the S protein. (B) Western blot analysis of the expressed His₆-tagged fragments. SF9, control SF9 cells; VA, fragment A; VB, fragment B; VC, fragment C; VD, fragment D; VE, fragment E; M, protein marker.

FIG. 3.

Representative immunoblot assays with sera from the 10 randomly selected SARS patients. Total cellular extracts of fragments A to E were subjected to SDS-12% PAGE and immunoblot analysis. For all of the immunoblot assays, sera were used at 1:100 and peroxidase-conjugated goat anti-total Ig polyclonal sera were used at 1:1,000. M, protein marker; SF9, cellular extracts from null-infected SF9 cells; A to E, cellular extracts from SF9 cells infected with VA, VB, VC, VD, and VE, respectively. (I) Immunoblot with one healthy human serum sample as a control. (II) Immunoblot with serum sample no. 1 that reacted with domains C, D, and E. (III) Immunoblot with serum sample no. 5 that recognized only the C domain of S protein. (IV) Immunoblot with serum sample no. 8 that recognized both the C and D domains.

FIG. 4.

Expression, purification, and immunoblot analysis of the His₆-tagged C domain from E. coli strain M15. (A) SDS-PAGE of samples eluted from affinity chromatography purification. M, protein marker; lane 1, total cellular extract before IPTG induction; lane 2, total cellular extract 4 h post-IPTG induction; lane 3, eluted protein sample from affinity chromatography purification. (B) Immunoblot of fragment C with the monoclonal anti-His₅ antibody. Lane 4, purified protein sample. (C) Immunoblot of purified fragment C with a pooled serum sample from the 10 positive serum samples. Lane 5, purified protein sample as described for lane 3.