Antigenic characterization of severe acute respiratory syndrome-coronavirus nucleocapsid protein expressed in insect cells: The effect of phosphorylation on immunoreactivity and specificity.
The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is involved in the pathological reaction to SARS and is a key antigen for the development of a sensitive diagnostic assay. However, the antigenic properties of this N protein are largely unknown. To facilitate the studies on the function and antigenicity of the SARS-CoV N protein, 6x histidine-tagged recombinant SARS-CoV N (rSARS-N) with a molecular mass of 46 and 48kDa was successfully produced using the recombinant baculovirus system in insect cells. The rSARS-N expressed in insect cells (BrSARS-N) showed remarkably higher specificity and immunoreactivity than rSARS-N expressed in E. coli (ErSARS-N). Most of all, BrSARS-N proteins were expressed as a highly phosphorylated form with a molecular mass of 48kDa, but ErSARS-N was a nonphosphorylated protein. In further analysis to determine the correlation between the phosphorylation and the antigenicity of SARS-N protein, dephosphorylated SARS-N protein treated with protein phosphatase 1 (PP1) remarkably enhanced the cross-reactivity against SARS negative serum and considerably reduced immunoreactivity with SARS-N mAb. These results suggest that the phosphorylation plays an important role in the immunoreactivity and specificity of SARS-N protein. Therefore, the BrSARS-N protein may be useful for the development of highly sensitive and specific assays to determine SARS infection and for further research of SARS-N pathology.
Shin
Gu-Choul
GC
Division of Influenza and Respiratory Viruses, Center for Infectious Disease, National Institute of Health, Korea Center for Disease Control and Prevention, 5 Nokbun-dong, Eunpyung-gu, Seoul 122-701, Republic of Korea.
Chung
Yoon-Seok
YS
Kim
In-Soo
IS
Cho
Hae-Wol
HW
Kang
Chun
C
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity / Q000502:physiology*; D000956:Antigens, Viral / Q000276:immunology*; D000086462:Coronavirus Nucleocapsid Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D007313:Insecta / Q000166:cytology; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010766:Phosphorylation; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins