AIM:Nucleocapsid (N) protein plays an important role inreproduction and pathological reaction of severe acuterespiratory syndrome (SARS) coronavirus (SCoV),theantigenicity of the protein is better than spike (S) protein.This study was to find a highly specific and antigenicrecombinant SCoV nucleocapsid (rSCoVN) protein,and toprovide a basis for further researches on early diagnosisof SARS.METHODS:Full length cDNA of SCoV nucleocapsid (SCoVN)protein was amplified through polymerase chain reaction(PCR) and cloned into yeast expression vector pPIC3.SKto construct plasmid of pPIC3.5K-SCoVN.The plasmidwas linearized and then transformed into Pichia pastoris(P.pastods) GSl15 (His’Mut~+) by electroporation.His~+Mut~+recombinant strains were identified by PCR and cultivatedon MM/MD plates.The influence of different factors onbiomass and rSCoVN protein production during inductionphase,such as various induction media,dissolved oxygen(DO) and different final concentrations of methanol,wassubsequently studied.The expression level and activationwere detected by SDS-PAGE and Western-blot respectively.RESULTS:All of the recombinants were His~+Mut~+ aftertransformation of P.pastoris with linearized plasmids.TheBMMY medium was optimal for recombinant ScoVN (rSCoVN)protein expression and growth of the recombinant strains.The final optimal concentration of methanol was 20 mL/L,the DO had a significant effect on rSCoVN protein expressionand growth of recombinant strains.The rSCoVN proteinexpressed in recombinant strains was about 8% of thetotal cell protein,520 mg/L of rSCoVN protein was achieved,and a maximum cell A at 600 nm of 62 was achieved inshake flask culture.The rSCoVN protein had a high specificityagainst mouse-anti-SARS-CoVN-mAb and SARS positivesera,but had no cross-reaction with normal human serum.The biological activity of rSCoVN expressed in P.pastoriswas about 4-fold higher than that expressed in E.co//whenthe same rSCoVN protein quantity was used.CONCLUSION:Active recombinant severe acute respiratory syndrome (SARS) coronavirus nudeocapsJd (rSCoVN) proteincan be successfully expressed in recombinant methylotrophicyeast P.pastoris GS115.The rSCoVN protein has a highspecificity against SARS-CoVN-mAb and SARS positivesera,but has no cross-reaction with normal human serum.This provides a basis for further researches on the earlydiagnosis of SARS and the mechanism of SCoV. AIM: Nucleocapsid (N) protein plays an important role in regeneration and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), theantigenicity of the protein is better than spike (S) protein. This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and toprovide a basis for further researches on early diagnosis of SARS. METHODS: Full length cDNA of SCoV nucleocapsid (SCoVN) protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.SKto construct plasmid of pPIC3.5K-SCoVN.The plasmidwas linearized and then transformed into Pichia pastoris (P.pastods) GS155 (His’Mut ~ +) by electroporation.His ~ + Mut ~ + recombinant strains were identified by PCR and cultivatedon MM / MD plates. The influence of different factors onbiomass and rSCoVN protein production during inductionphase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, wassubseque was studied. The expression level and activationwere detected by SDS-PAGE and Western-blot respectively .RESULTS: All of the recombinants were His ~ + Mut ~ + aftertransformation of P. pastoris with linearized plasmids.The BMMMY medium was optimal for recombinant ScoVN (rSCoVN ) protein expression and growth of the recombinant strains. The final optimal concentration of methanol was 20 mL / L, the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN proteinexpressed in recombinant strain was about 8% of the total cells protein, 520 mg / L of rSCoVN protein was achieved, and a maximum cell A at 600 nm of 62 was achieved inshake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum. The biological activity of rSCoVN expressed in P. pastoriswas about 4-fold higher than that expressed in E. co // whenthe same rSCoVN protein quantity was used. CONCLUSION: Active recombinant severe acute respiratory syndrome (SARS) coronavirus nudeocaps Jd (rSCoVN) proteincan be successfully expressed in recombinant methylotrophic pestal sulfate P. pastoris GS115. The rSCoVN protein has a high specificity against SARS-CoVN-mAb and SARS positive sera, but has no cross-reaction with normal human serum. This provides a basis for further researches on the early diagnosis of SARS and the mechanism of SCoV.