Expression, Antigenicity Identification And DNA Immunization Of SARS-CoV Structural Proteins

The severe acute respiratory syndrome (SARS), also named infectious atypical pneumonia, is a newly described and highly contagious respiratory infection that firstly occurred in late 2002 in Guangdong Province, China, and spread to more than 30 countries in early 2003. In March 2003, a novel coronavirus was identified as the etiological agent of SARS, which was termed as SARS-CoV. The coronaviruses are a diverse group of large, enveloped viruses that cause respiratory and enteric diseases in humans and other animals. The genome of SARS-CoV is a 29,727-nucleotide, positive-strand RNA, has typical gene order of coronaviruses [5'-replicase (rep), spike (S), envelope (E), Membrane (M), nucleotide (N)-3'] and short untranslated regions at both termini. The structural proteins of SARS-CoV, including S, E, M, and N, are common to all known coronaviruses. In the present study, the four structural proteins of SARS-CoV were expressed in E.coli and were used to screened the sera of convalescence phase of SARS patients for the antibodies against SARS-CoV. The S and N protein DNA vaccine were constructed and were used to immunized mice. The results may be helpful for understanding the immunological characteristic of SARS-CoV and developing the method for prophylaxis and treatment against SARS-CoV infection.1. Expression of the structural proteins of SARS-CoV in E.coli and analysis of antibodies against SARS-CoV in the sera of convalescence phase of SARS patientsThe full length sequences of E, M, N, 3CL gene, and four fragments of S gene were synthesized according to the published SARS-CoV Tor2 isolate. The fragments of S gene encode amino acid (aa) 14-313(S300), 301-540(S240), 557-894(S338) and 867-1190(S324) of S protein, respectively. The DNA fragments were inserted into prokaryotic expression plasmid pBV220, pPROEX HTa, pET-21a, and pGEX-4T-l. The recombinant expression plasmids were transformed into suitable host E.coli, The expression of the interest proteins were induced by heating or IPTG and analyzed by SDS-PAGE and Western Blot analysis. The pooled sera of convalescence phase of two SARS patients was used as the capture antibody. The SDS-PAGE analysis showed that the bacteria that transformed with the S300, S240, S338, S324, E, M, N and 3CL expression plasmid could express the proteins with the predicted molecular weight. Western Blot analysis showed that the binding reactivity of N protein is much more active, followed by S protein, and that of M protein is inferior. In the four fragments of S protein, the binding reactivity of S300 and S240 at N-terminal of S protein is more powerful than that of S338 and S324. The E and 3CL protein did notdevelop obvious binding reactivity. The nature of expression by E.coli may influence the antigenicity of the proteins, but the results could indicate that the N protein was the more active for induction of antibodies than S, M and E protein, and the main antigenic determinants of S protein locate at its N-terminal.N422, S300 and S240 protein was purified by Ni-NTA affinity resin and was used to coated the microtiter plates for detection of the anti- SARS-CoV IgG antibodies. In 30 sera samples of convalescence phase of patients clinically diagnosed SARS, there were 25 were positive for anti-N IgG, 15 were positive for anti-S300 IgG, and 12 were positive for anti-S240 IgG. The results further indicated that N protein is more sensitive for diagnosis for SARS. It is reported that the S protein of other coronavirus were of high antigenic and the antibodies against this protein is protective. If this do so for SARS-CoV, the result implied that the role of S antibodies in SARS-CoV infection is still to be further investigated and the re-infection of SARS-CoV may be occur.2. Immune responses induced by DNA vaccine of S and N protein in miceThe wild-type DNA sequence encoding aa 1-251 of S protein was amplified by PCR and the modified DNA sequence was synthesized using optimized codons from highly expressed mammalian genes. With EGFP fusion gene expression system, the syntheti...