Identification of Epitopes on SARS-CoV Nucleocapsid Protein that Induce the Cross-or Specific-Reactivity among SARS-CoV,HCoV-OC43 and HCoV-229E
To determine the specific epitopes on SARS-CoV nucleocapsid(N) protein,the cross-reactivities among three human coronavirus N proteins were analyzed systematically.The vaccinia recombinants were constructed to express N proteins of SARS-CoV,HCoV-OC43 and HCoV-229E.The polyclonal antibodies against the expressed N proteins were prepared with the correlated vaccinia recombinants.The indirect immunofluorescence was used to detect both the expression of N proteins in vaccinia recombinants and the cross-reactivity with antisera from three HCoVs(SARS-CoV,HCoV-OC43 and HCoV-229E) and SARS convalescent patient.The cross-or specificreactive epitopes on N proteins of three HCoVs above were further analyzed by Western blot with the E.coli expressed 39 peptides overlapping the whole N protein of SARS-CoV and also with the 4 antisera above.Immunofluorescence indicated the well expression of N proteins of SARS-CoV,HCoV-OC43 and HCoV-229E in vaccinia recombinant infected-HeLa cells and the clear antigenic cross-reactivity among the three N proteins.Four dominant antigenic epitopes on nucleocapsid protein of SARS-CoV were identified by Western blot,which were located at the amino acid positions of 30-60aa,170-184aa,301-320aa and 360-422aa.The epitopes which were cross-reactive between SARS-CoV and HCoV-OC43 were at 30-60aa,90-120aa,204-214aa and 320-360aa.The epitopes which were cross-reactive between SARS-CoV and HCoV-229E were at 30-60aa,150-160aa and 301-360aa.Importantly,we found that recombinant peptides N155b(60-214aa) and N185(30-214aa) containing the specific epitopes for SARS-CoV N protein were only reactive with the sera from SARS convalescent patient and mouse antiserum immunized with inactivated SARS-CoV,but not reactive with the goat anti-serum prepared with inactivated HCoV-OC43 or HCoV-229E.The results above provided very useful information for the development of specific diagnostic reagent using the recombinant SARS-CoV N protein.