Title
Biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and 229E human coronaviruses
Author
Tswen‐Kei Tang, Mark P.‐J. Wu, Shui‐Tsung Chen, Ming‐Hon Hou, Ming‐Hsiang Hong, Fu‐Ming Pan, Hui‐Ming Yu, Jenn‐Han Chen, Chen‐Wen Yao, Andrew H.‐J. Wang
Year
2005
Journal
Proteomics
Abstract
Severe acute respiratory syndrome (SARS) is a serious health threat and its early diagnosis is important for infection control and potential treatment of the disease. Diagnostic tools require rapid and accurate methods, of which a capture ELISA method may be useful. Toward this goal, we have prepared and characterized soluble full‐length nucleocapsid proteins (N protein) from SARS and 229E human coronaviruses. N proteins form oligomers, mostly as dimers at low concentration. These two N proteins degrade rapidly upon storage and the major degraded N protein is the C‐terminal fragment of amino acid (aa) 169–422. Taken together with other data, we suggest that N protein is a two‐domain protein, with the N‐terminal aa 50–150 as the RNA‐binding domain and the C‐terminal aa 169–422 as the dimerization domain. Polyclonal antibodies against the SARS N protein have been produced and the strong binding sites of the anti‐nucleocapsid protein (NP) antibodies produced were mapped to aa 1–20, aa 150–170 and aa 390–410. These sites are generally consistent with those mapped by sera obtained from SARS patients. The SARS anti‐NP antibody was able to clearly detect SARS virus grown in Vero E6 cells and did not cross‐react with the NP from the human coronavirus 229E. We have predicted several antigenic sites (15–20 amino acids) of S, M and N proteins and produced antibodies against those peptides, some of which could be recognized by sera obtained from SARS patients. Antibodies against the NP peptides could detect the cognate N protein clearly. Further refinement of these antibodies, particularly large‐scale production of monoclonal antibodies, could lead to the development of useful diagnostic kits for diseases associated with SARS and other human coronaviruses.
Instrument
J-720
Keywords
Circular dichroism, Secondary structure, Biochemistry