Expression and purification of recombinant nucleocapsid protein of SARS Coronavirus and preparation of its monoclonal antibodies

To express and purify the nucleocapsid protein(N protein) of SARS Coronavirus(SARS-CoV) and to generate monoclonal antibodies with high security and specificity against N protein,genes encoding N protein were separated into two parts according to the prediction of antigenicty,the first part designed from 1 to 549 base pairs(N-terminus),the second from 496 to 1269 base pairs(C-terminus).They were obtained by nucleotide synthesis and were cloned into expression vector pET32a(+) respectively.Proteins then were induced to expression by IPTG and purified through Ni-NTA.Balb/c mice were immunized with purified recombinant protein to prepare McAb by hybridoma technique and the McAb was selected by indirect ELISA.The specificity of McAb was identified by Western blot and indirect fluorescence assay.Results showed that target proteins were successfully expressed and purified and seven hybridoma cell lines secreting monoclonal antibodies against SARS-CoV N1 and two hybridoma cell lines secreting monoclonal antibodies against SARS-CoV N2 were obtained.About the immunoglobulin subclass of McAb,six were IgG1,two were IgG2b and one was IgG3.Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with SARS-CoV N protein.It is concluded that the recombinant SARS-CoV N protein were expressed and purified and nine specific McAb against SARS-CoV N protein were obtained successfully in the present study.