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Spike protein is one of the major structural proteins of severe acute respiratory syndrome-coronavirus. It is essential for the interaction of the virons with host cell receptors and subsequent fusion of the viral envelop with host cell membrane to allow infection. Some spike proteins of coronavirus, such as MHV, HCoV-OC43, AIBV and BcoV, are proteolytically cleaved into two subunits, S1 and S2. In contrast, TGV, FIPV and HCoV-229E are not. Many studies have shown that the cleavage of spike protein seriously affects its function. In order to investigate the maturation and proteolytic processing of the S protein of SARS CoV, we generated S1 and S2 subunit specific antibodies (Abs) as well as N, E and 3CL protein-specific Abs. Our results showed that the antibodies could efficiently and specifically bind to their corresponding proteins from E.coli expressed or lysate of SARS-CoV infected Vero-E6 cells by Western blot analysis. Furthermore, the anti-S1 and S2 Abs were proved to be capable of binding to SARS CoV under electron microscope observation. When S2 Ab was used to perform immune precipitation with lysate of SARS-CoV infected cells, a cleaved S2 fragment was detected with S2-specific mAb by Western blot analysis. The data demonstrated that the cleavage of S protein was observed in the lysate, indicating that proteolytic processing of S protein is present in host cells.

The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (N1 and N2) of the N protein of SARS-CoV were predicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodies were isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-induced polyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SARS-CoV. Furthermore, it was confirmed that N1 peptide-specific IgG antibodies were detectable in the sera of severe acute respiratory syndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified and N protein specific Abs were produced by peptide immunization, which will be usefull for the study of SARS-CoV.

Prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein expressed predominantly in prostate secretory acinar epithelium and prostate cancer cells as well as in several extraprostatic tissues. Mouse monoclonal antibody 4G5 specific to the extracellular domain of PSMA was used to screen two phage displayed peptide libraries (9aa linear and 9aa cys library). Three 4G5-reactive phagotopes were identified. Sequence analysis of isolated clones demonstrated that the interaction motif "VDPA/SK" has high homology to 719-725aa on PSMA. Immunohistochemical staining of the prostate cancer sample with the PSMA-mimic phagotope (mimotope) immunized serum antibodies demonstrate that the mimotope isolated from the phage displayed peptide libraries can induce PSMA specific immune response in vivo.

Trichosanthin (TCS) is a potent allergen to mice. According to our previous experiments, it could bring out the IgE response to ovabumin (OVA) if TCS was given one day before OVA immunization, while OVA alone could not induce IgE to it. In this work, the kinetics of interleukin 4(IL-4) and interferon gamma(IFN-gamma) gene expression in the mesenteric lymph node (MLN) of TCS-immunized mice was investigated using a semi-quantitative RT-PCR method. It indicated that TCS induced significant IL-4 gene expression and the peaks of IL4 gene expression were on day one after TCS immunization in both primary and secondary response. In contrast, the IFN-gamma gene expression was suppressed. Furthermore, the IL-4 gene expression in the secondary response was lower than that in the primary response. Thus the presence of IgE memory B cells were studied. Results showed that the amount of mature IgE mRNA arose significantly and rapidly one day after TCS restimulation, while in the MLN of the mice primed 30 days before and without boost, it was almost as the same amount of the unimmunized control. These findings suggest the existence of the IgE memory B cells in the mice after the primary TCS immunization.

The antigenic determinants of trichosanthin were predicted by molecular modeling. First, the three-dimensional structure model of the antigen-binding fragment of anti-trichosanthin immunoglobulin E was built on the basis of its amino-acid sequence and the known three-dimensional structure of an antibody with similar sequence. Secondly, the preferable antigen-antibody interactions were obtained based on the known three-dimensional structure of trichosanthin and of the hypervariable regions of anti-trichosanthin immunoglobulin E. Two regions in the molecular surface of trichosanthin were found to form extensive interactions with the hypervariable regions of the antibody and have been predicted to be the possible antigenic determinants: one is composed of two polypeptide segments, Ile201-Glu210 and Ile225-Asp229, which are close to each other in the three-dimensional structure; and the other is the segment Lys173-Thr178. The former region seems to be the more reasonable antigenic determinant than the latter one.

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important viral structural protein. Based on bioinformatics analysis, 10 antigenic peptides derived from the S protein sequence were selected and synthesized. The antigenicity and immunoreactivity of all the peptides were tested in vivo and in vitro. Four peptides (P6, P8, P9 and P10) which contain B cell epitopes of the S protein were identified, and P8 peptide was confirmed in vivo to have a potential in serological diagnosis. By using a syncytia formation model, we tested the neutralization ability of all 10 peptides and their corresponding antibodies. It is interesting to find that P8 and P9 peptides inhibited syncytia formation, suggesting that the P8 and P9 spanning regions may provide a good target for anti-SARS-CoV drug design. Our data suggest that we have identified peptides derived from the S protein of SARS-CoV, which are useful for SARS treatment and diagnosis.