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
Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods:...Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods: The recombinant baculovirus bac-S0.7 with the 700 bp fragment of S gene 5' terminal of Hantaan virus was constructed, and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay, micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0.7 infecting insect cells, specific antibody with the highest titer of 1∶1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0.7 gene fragment in insect cells is immunogenic.展开更多
To characterize the antigenicity of nucleocapsid proteins(NP) derived from canine coronavirus(CCo V) and canine respiratory coronavirus(CRCo V) in China, the N genes of CCo V(CCo V-BJ70) and CRCo V(CRCo V-BJ202) were ...To characterize the antigenicity of nucleocapsid proteins(NP) derived from canine coronavirus(CCo V) and canine respiratory coronavirus(CRCo V) in China, the N genes of CCo V(CCo V-BJ70) and CRCo V(CRCo V-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs(r NPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCo V-BJ70 and CRCo V-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) results showed that r NPs of CCo V and CRCo V were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the r NP from CRCo V could cross-react with mice antisera against human coronavirus(HCo V-229 E, NL63, OC43, HKU1), while r NP of CCo V had cross-reactivity with only anti-sera against viruses belonging to the same group(HCo V-229 E and NL63). In summary, CCo V and CRCo V r NPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.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.
基金National Natural Science Foundation of China (No.30070686)Chinese Educational Deputy Fund for skeleton teachers
文摘Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods: The recombinant baculovirus bac-S0.7 with the 700 bp fragment of S gene 5' terminal of Hantaan virus was constructed, and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay, micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0.7 infecting insect cells, specific antibody with the highest titer of 1∶1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0.7 gene fragment in insect cells is immunogenic.
基金supported by the Mega project for Infectious Disease Research of China (2014ZX10004001-002, 2013ZX10004601)National Basic Research Program of China (2011CB504704)
文摘To characterize the antigenicity of nucleocapsid proteins(NP) derived from canine coronavirus(CCo V) and canine respiratory coronavirus(CRCo V) in China, the N genes of CCo V(CCo V-BJ70) and CRCo V(CRCo V-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs(r NPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCo V-BJ70 and CRCo V-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) results showed that r NPs of CCo V and CRCo V were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the r NP from CRCo V could cross-react with mice antisera against human coronavirus(HCo V-229 E, NL63, OC43, HKU1), while r NP of CCo V had cross-reactivity with only anti-sera against viruses belonging to the same group(HCo V-229 E and NL63). In summary, CCo V and CRCo V r NPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.
