Studies Of Antigenicity Identification, DNA Immunization And RNA Interference Of SARS-CoV Structural Proteins

The infectious atypical pneumonia, also named severe acute respiratory syndrome (SARS), 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 immunize mice. Several small interference RNAs (siRNA) were identified that could inhibit the expression of N and S gene efficiently. The results may be helpful for understanding the immunological characteristic of SARS-CoV and developing the methods for prophylaxis and treatment against SARS-CoV infection.1, Expression and antigeniciry identification of the structural proteins of SARS-CoVThe full length sequence of E, N, 3CLpro gene, two fragments of M gene, and four fragments of S gene were synthesized according to the published sequence of SARS-CoV. The DNA fragments were inserted into prokaryotic expression plasmid pPROEX HT, pBV220, pET21a or pGEX-4T-l. The recombinant expression plasmids were transformed into suitable host E.coli. The expression of the interest proteins was induced and analyzed. The SDS-PAGE analysis showed that the bacteria that transformed with the S300 (aa 14-313 of S protein), S240 (aa 301-540 of S protein), S338 (aa 557-894 of S protein), S324 (aa 867-1190 of S protein), E, MC (aa 111-221 of M protein), N and 3 CLpro expression plasmid could express the proteins with the predicted molecular weight. These expression products were subjected to Western Blot analysis with pooled sera of convalescence phase of two SARS patients. The results showed that the binding reactivity of N protein is much more active, followed by S protein, and that of MC protein is inferior. In the four fragments of S protein, the binding reactivity of S300 at N-terminal of S protein is stronger than that of S240 and S338, and the reactivity of S324 is somewhat weak. The E and 3CLpro did not develop obvious binding reactivity. The results indicated to some extent that the N protein was more powerful for induction of antibodies than S, M protein, and the main antigenic determinants of S protein locate at its N-terminal.S300, S240 and N protein were purified by Ni-NTA, respectively, and were used to coated the microtiter plates for detection of the anti-SARS-CoV IgG antibodies. In 18 sera samples of convalescence phase of patients clinically diagnosed SARS, there were 17 were positive for anti-NIgG, 14 were positive for anti-S300 IgG, and 10 were positive for anti-S240 IgG, respectively. The results further indicated that the N protein is more sensitive for diagnosis for SARS-CoV infection. The results also suggested that the role of S antibodies in SARS-CoV infection is still to be further investigated.2, Immune responses induced by DNA vaccines of S and N proteins in miceThe modified DNA sequence encoding aa 1-248 of S protein was synthesized using optimized codons from highly expressed mammalian genes. With EGFP fusion gene expression system, the synthetic DNA sequence was confirmed to show extremely higher expression efficiency than the wild-type sequence. The DNA sequence encoding nt 1-2058 and nt 1913-3768 of S gene were amplified and spliced by PCR, respectively, then the full length S gene was...