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. 2006 Oct;33(2):147-56.
doi: 10.1007/s11262-005-0050-8.

Identification of a B-cell antigenic epitope at the N-terminus of SARS-CoV M protein and characterization of monoclonal antibody against the protein

Chao Qian  1 , Di Qin, Qiao Tang, Yi Zeng, Guixia Tang, Chun Lu
Affiliations

Identification of a B-cell antigenic epitope at the N-terminus of SARS-CoV M protein and characterization of monoclonal antibody against the protein

Chao Qian et al. Virus Genes. 2006 Oct.

Abstract

To identify the potential B-cell antigenic epitopes within the N-terminus of SARS-CoV (SARS-associated coronavirus, SARS-CoV) M protein and characterize monoclonal antibody (MAb) against the protein as well as its recognizing region, we expressed and purified a portion of SARS-CoV M protein (amino acid 1-43) in Escherichia coli (E. coli). By using Western blot and enzyme-linked immunosorbent assay (ELISA), we showed that the purified recombinant M protein could be recognized by four SARS-CoV-positive human sera even when those sera were 12,800-fold diluted. Furthermore, we characterized one representative IgG2 MAb, 3H9, which exhibited a strong immunoreaction to both recombinant M protein and native viral protein of SARS-CoV. We found a B-cell antigenic epitope located between amino acid 1-15 and defined the MAb recognizing region within amino acid 16-28 of M. These findings not only suggest that both recombinant M protein and its specific MAbs may be used as the diagnostic reagents for SARS, but also provide a potential target site for the design of an epitope-based vaccine against SARS.

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Figures

Fig. 1
Fig. 1
SDS-PAGE analysis on expression and purity of M protein in E. coli BL21. (a) SDS-PAGE analysis of expression of M protein in E. coli BL21. E. coli BL21 was transformed with the recombinant plasmid pSARS-M, followed by induction with IPTG. The bacterial cultural crude materials were subjected to the 10% SDS-PAGE. Lane 1: molecular weight marker; Lane 2: cellular extracts of TPTG-induced BL21 transformed with the empty plasmid pGEX-6P-1 (control); Lane 3: cellular extracts of IPTG-induced BL2 1 transformed with the recombinant pSARS-M construct. (b) SDS-PAGE analysis of purity of recombinant M protein after purified by affinity chromatography. The crude material of BL21 transformed with pSARS-M plasmid was subjected to sonication. The supernatants were filtered through a 0.45 μm nitrocellulose membrane and loaded onto Glutathione Sepharose 4B affinity chromotography column. The purified proteins were detected by SDS-PAGE. Lane 1: molecular weight marker; Lane 2: total cellular extracts of IPTG-induced BL21 (control); Lane 3: total cellular extracts of IPTG-induced BL21 transformed with the recombinant pSARS-M construct (control); Lane 4: eluted protein of IPTG-induced BL21 transformed with the recombinant pSARS-M construct from affinity chromatography purification. Arrows indicate M fusion protein expressed in E coli BL21 (left) and purified M fusion protein (right). The gel was stained by Coomassie blue. The molecular mass size markers (in KDa) are reported on the left of each Figure
Fig. 2
Fig. 2
Analysis of antigenicity of expressed M protein. (a) Western blot analysis on the purified M fusion protein with the sera of SARS-CoV-positive patients. M protein was purified from lysates of BL21 cells transformed with pSARS-M plasmid through Glutathione Sepharose 4B affinity chromotography column. Purified proteins were subjected to SDS-PAGE and subsequently transferred to PVDF membrane for Western blot with the sera of SARS-CoV-positive patients. Lane 1: eluted protein of IPTG-induced BL21 transformed with the empty plasmid pGEX-6P-1 (control); Lane 2: eluted protein of IPTG-induced BL21 transformed with the recombinant pSARS-M construct. Results are Western blot probed with the serum from sample 1. Additional three experiments with the sera from sample 2, 3 and 4, respectively, were also performed and gave similar results. The properties of sera from four SARS-CoV-positive patients were listed in Table 1. Arrow indicates purified M fusion protein immunoblotting with the serum of SARS-CoV-positive patient. (b) Detection of eight human sera by indirect ELISA by using purified M fusion protein as antigen. The purified recombinant M protein was diluted and used to coat 96-well plates. SARS-CoV-positive human serum diluted in a twofold series from 1:100 to 1:12,800 was added into each well in duplicate. The OD value was read at 450 nm in CliniBio 128C reader and the cut-off value was defined as the mean OD plus 2.1 standard deviations calculated from the four negative samples used as controls. Sample 1–4 were sera from four confirmed SARS patients and 5–8 from four healthy people (controls), whose source and properties were listed in Table 1
Fig. 3
Fig. 3
Localization of B-cell antigenic epitope in M protein. (a) SDS-PAGE analysis of truncated M protein expressed in E. coli BL21. BL21 was transformed with two truncated recombinant M protein-coding genes constructs, followed by induction with IPTG. The bacterial cultural crude materials were detected by the 10% SDS-PAGE and then stained with Coomassie blue. Lane 1: molecular weight marker; Lane 2: cellular extracts of IPTG-induced BL21 transformed with the recombinant pSARS-M construct (control); Lane 3: cellular extracts of IPTG-induced BL21 transformed with the recombinant pSARS-Ml construct; Lane 4: cellular extracts of IPTG-induced BL21 transformed with the recombinant pSARS-M2 construct. (b) Localization of B-cell antigenic epitope in M protein by Western blot with the sera of SARS-CoV-positive patients. The crude materials of inductive culture of E. coli BL21 were subjected to SDS-PAGE and subsequently transferred to PVDF membrane for Western blot analysis with the SARS-CoV-positive human serum. Lane 1: total cellular extracts of IPTG-induced BL21 transformed with pSARS-M2 construct; Lane 2: total cellular extracts of IPTG-induced BL21 transformed with pSARS M1 construct; Lane 3: total cellular extracts of IPTG-induced BL21 transformed with pSARS-M construct (control). A representative experiment is shown with the serum from sample 1. Additional three experiments with the sera from sample 2, 3 and 4, respectively, were also performed and gave similar results. The properties of sera from four-SARS-CoV-positive patients were listed in Table 1
Fig. 4
Fig. 4
Identification of the MAb specificity. (a) Western blot analysis of the expressed M fusion protein with the MAb, 3H9. The crude materials of inductive culture of E. coli BL21 were subjected to SDS-PAGE and subsequently transferred to PVDF membrane for Western blot with the MAb, 3H9. Lane 1: total cellular extracts of IPTG-induced BL21 (control); Lane 2: total cellular extracts of IPTG-induced BL21 transformed with pGEX-6p-l (control); Lane 3: total cellular extracts of IPTG-induced BL21 transformed with the recombinant pSARS-M construct. Arrow indicates expressed M fusion protein immunoblotting with the MAb. (b) ELISA reactivity of the MAb, 3H9, to viral protein of SARS-CoV. A total of 96-well plates from the diagnostic kit detecting antibody specific to SARS virus, which were coated with the UV-irradiated and extracted viral protein of SARS-CoV, were used to determine the titers of MAb in the supernatants and ascites fluid by indirect ELISA. Supernatants and ascites fluid from 3H9 and S/P2.0 (controls) diluted in a two-fold series from 1:100 to 1:6,400 were added into each well in duplicate, respectively. The OD value was read at 450 nm in CliniBio 128C reader and the cut-off value was defined as the mean OD plus 2.1 standard deviations calculated from the four negative samples used as controls
Fig. 5
Fig. 5
Localization of the recognizing region of the MAb, 3H9 in recombinant M protein by Western blot. The crude materials of E. coli BL21 culture were subjected to SDS-PAGE and subsequently transferred to PVDF membrane for Western blot with the MAb, 3H9. Lane 1: total cellular extracts from IPTG-induced BL21 transformed with recombinant pSARS-M construct (control); Lane 2: total cellular extracts from IPTG-induced BL21 transformed with recombinant pSARS-Ml construct; Lane 3: total cellular extracts from IPTG-induced BL21 transformed with recombinant pSARS-M2 construct
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