Cloning, expressing and antigenicity analysis of nucleocapsid proteins of SARS-CoV, HCoV-229E and OC43
Objective To obtain a recombinant nucleocapsid(N) protein of SARS-CoV, HCoV-229E and HCoV-OC43, respectively and to study antigenic relationships of N proteins between SARS-CoV and human coronaviruses 229E and OC43. Methods The genes encoding the full-length of N proteins from SARS-CoV, HCoV-229E and HCoV-OC43 were amplified by RT-PCR and cloned into the prokaryotic expression vector pQE30. The His6-tagged N proteins were expressed in the M15 strain and further purified with affinity chromatography. The antigenicity of N proteins was analyzed by Western blot and Immunofluorescence assay. Results The N genes of 1281, 1182 and 1359 bp from SARS-CoV, HCoV-229E and HCoV-OC43, respectively were amplified with their corresponding primer pairs. The recombinant plasmids were sequenced, and they were all in frame with sequences matching those for the N genes of the three coronaviruses. The expressed recombinant His6-tagged N proteins were identified by Western blot assay with anti-His tag monoclonal antibody. The immunoreactive protein bands with expected sizes were 47 kDa, 44 kDa and 50 kDa from SARS-CoV, HCoV-229E and HCoV-OC43, respectively. The nucleocapsid proteins of SARS-CoV, HCoV-229E and HCoV-OC43 strongly and specifically reacted with the virus specific rabbit serum and with the nucleoprotein specific murine serum. No cross-reactivity was found among the nucleocapsid proteins of SARS-CoV, HCoV-229E and HCoV-OC43. Conclusion The immunogenic nucleocapsid recombinant proteins from SARS-CoV, HCoV-229E and HCoV-OC43 were obtained. There was no antigenic relationship among the three N proteins.