The nucleocapsid (N) protein of SARS-coronavirus (SARS-CoV) is the key protein for the formation of the helical nucleocapsid during virion assembly. This protein is believed to be more conserved than other proteins of the virus, such as spike and membrane glycoprotein. In this study, the N protein of SARS-CoV was expressed in Escherichia coli DH5ฮฑ and identified with pooled sera from patients in the convalescence phase of SARS. A plasmid pCI-N, encoding the full-length N gene of SARS-CoV, was constructed. Expression of the N protein was observed in COS1 cells following transfection with pCI-N. The immune responses induced by intramuscular immunization with pCI-N were evaluated in a murine model. Serum anti-N immunoglobulins and splenocytes proliferative responses against N protein were observed in immunized BALB/c mice. The major immunoglobulin G subclass recognizing N protein was immunoglobulin G2a, and stimulated splenocytes secreted high levels of gamma interferon and IL-2 in response to N protein. More importantly, the immunized mice produced strong delayed-type hypersensitivity (DTH) and CD8 + CTL responses to N protein. The study shows that N protein of SARS-CoV not only is an important B cell immunogen, but also can elicit broad-based cellular immune responses. The results indicate that the N protein may be of potential value in vaccine development for specific prophylaxis and treatment against SARS. ยฉ 2004 Elsevier Inc.