Antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein

Abstract

Background: The widespread threat of severe acute respiratory syndrome (SARS) to human health has made urgent the development of fast and accurate analytical methods for its early diagnosis and a safe and efficient antiviral vaccine for preventive use. For this purpose, we investigated the antigenicity of different regions of the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein.

Methods: The cDNA for full-length N protein and its various regions from the SARS-CoV was cloned and expressed in Escherichia coli. After purification, all of the protein fragments were printed on glass slides to fabricate a protein microarray and then probed with the sera from SARS patients to determine the reactivity of these protein fragments.

Results: The full-length protein and two other fragments reacted with all 52 sera tested. Four important regions with possible epitopes were identified and named as EP1 (amino acids 51-71), EP2 (134-208), EP3 (249-273), and EP4 (349-422), respectively. EP2 and EP4 possessed linear epitopes, whereas EP1 and EP2 were able to form conformational epitopes that could react with most (>80%) of the tested sera. EP3 and EP4 also formed conformational epitopes, and antibodies against these epitopes existed in all 52 of the sera tested.

Conclusion: The N protein is a highly immunogenic protein of the SARS-CoV. Conformational epitopes are important for this protein, and antigenicity of the COOH terminus is higher than that of the NH(2) terminus. The N protein is a potential diagnostic antigen and vaccine candidate for SARS-CoV.

Figure 1.

Positions of the fragments on the SARS-CoV N protein. The line with scales at the top represents the full-length N protein (N4), with the numbers indicating amino acid positions. The other lines represent the fragments, and the numbers on both ends of each fragment indicate the starting and ending amino acid positions. The fragment name is above the line; the numbers in parentheses indicate the percentages of sera positive for each fragment by protein microarray analysis. The four important epitope regions (EP1–EP4) are located at amino acids 51–71, 134–208, 249–273, and 349–422, respectively, as indicated by the thick lines.

Figure 2.

Correlation between FI and captured IgG. (A), correlation between FI and concentration of printed IgG. Human IgG was printed in increasing concentrations and probed with Cy5-labeled goat anti-human IgG. The FI was plotted as a function of the concentration of printed human IgG to generate a dose–response curve, and a good correlation coefficient (close to 1) was obtained. (B), correlation between FI and amount of printed human IgG.

Figure 3.

Reactivities of all of fragments printed at the concentration of 400 mg/L. The fluorescence values were normalized and logarithmically transformed (base 2); the cutoff value 8 was subtracted from each result. The values were then clustered by CLUSTER software and viewed with TREEVIEW software. Black squares represent positive values, white squares indicate negative values, and gray squares indicate intermediate degrees of FI.