The nueleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (Nl and N2) of the N protein of SARS-CoV were predicted by bio...The nueleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (Nl 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 SAR,S-CoV. Furthermore, it was confirmed that Nl 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 useful for the study of SARS-CoV.展开更多
Thichosanthin (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 no...Thichosanthin (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 γ(IFN-γ) 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-4gene 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-γ 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 rpemory 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.展开更多
To prepare monoclonal antibody specific to epidermal growth factor receptor(EGFR)intracellular domain,its gene was amplified from total RNA of A431 cell by RT-PCR.Then the gene was cloned into prokaryotic vector pET30...To prepare monoclonal antibody specific to epidermal growth factor receptor(EGFR)intracellular domain,its gene was amplified from total RNA of A431 cell by RT-PCR.Then the gene was cloned into prokaryotic vector pET30a(+).The recombinant plasmid was transformed into E.coli BL21(DE3)strain for protein expression. Recombinant protein was induced with IPTG and purified using Ni^(2+)NTA agarose.Then the anti-EGFR monoclonal antibody(mAb)was prepared with classical hybridoma technique.Positive clones were selected using indirect enzyme-linked immunoabsorbent assay(ELISA).Totally 4 hybridoma clones were obtained and these mAbs were IgGl(3 clones)and IgG2a(I clone),respectively.Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line.The mAbs will be useful in the study of EGFR-mediated signal transduction.Cellular & Molecular Immunology.2004;1(2):137-141.展开更多
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 en... 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, S 1 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 S 1 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-S 1 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.展开更多
基金supported by the grant of Shanghai Science and Technology Committee(No.03DZ19113)National Key Basic Research Program of China(No.2001CB510006)+1 种基金863 project(No.2001AA231011)a specific project against SARS from Chinese Academy of Sciences.
文摘The nueleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (Nl 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 SAR,S-CoV. Furthermore, it was confirmed that Nl 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 useful for the study of SARS-CoV.
文摘Thichosanthin (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 γ(IFN-γ) 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-4gene 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-γ 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 rpemory 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.
基金supported by Science and Technology Commission of Shanghai Municipality(03JC14085)grant of National Natural Science Foundation#30170888the grant from E-institutes of Shanghai Universities Immunology Division.
文摘To prepare monoclonal antibody specific to epidermal growth factor receptor(EGFR)intracellular domain,its gene was amplified from total RNA of A431 cell by RT-PCR.Then the gene was cloned into prokaryotic vector pET30a(+).The recombinant plasmid was transformed into E.coli BL21(DE3)strain for protein expression. Recombinant protein was induced with IPTG and purified using Ni^(2+)NTA agarose.Then the anti-EGFR monoclonal antibody(mAb)was prepared with classical hybridoma technique.Positive clones were selected using indirect enzyme-linked immunoabsorbent assay(ELISA).Totally 4 hybridoma clones were obtained and these mAbs were IgGl(3 clones)and IgG2a(I clone),respectively.Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line.The mAbs will be useful in the study of EGFR-mediated signal transduction.Cellular & Molecular Immunology.2004;1(2):137-141.
文摘 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, S 1 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 S 1 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-S 1 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.
