[Molecular cloning and expression of the severe acute respiratory syndrome-associated coronavirus nucleocapsid protein and its clinical application].
OBJECTIVE
To clone and express nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus, and to evaluate its antigenicity and application value in the development of serological diagnostic test for SARS.


METHODS
SARS-associated coronavirus N protein gene was amplified from its genomic RNA by reverse transcript nested polymerase chain reaction (RT-nested-PCR) and cloned into pBAD/Thio-TOPO prokaryotic expression vector. The recombinant N fusion protein was expressed and purified, and its antigenicity and specificity was analyzed by Western Blot, to establish the recombinant N protein-based ELISA for detection of IgG antibodies to SARS-associated coronavirus, and SARS-associated coronavirus lysates-based ELISA was compared parallelly.


RESULTS
The recombinant expression vector produced high level of the N fusion protein after induction, and that protein was purified successfully by affinity chromatography and displayed higher antigenicity and specificity as compared with whole virus lysates.


CONCLUSIONS
The recombinant SARS-associated coronavirus N protein possessed better antigenicity and specificity and could be employed to establish a new, sensitive, and specific ELISA for SARS diagnosis.
Lu
Jian
J
Shenzhen Donghu Hospital, Shenzhen, China.
Zhou
Bai-ping
BP
Zhou
Yu-sen
YS
Jiang
Xiao-ling
XL
Wen
Li-xia
LX
Le
Xiao-hua
XH
Li
Bing
B
Xu
Liu-mei
LM
Li
Li-xiong
LX
D000914:Antibodies, Viral / Q000097:blood; D015153:Blotting, Western; D003001:Cloning, Molecular; D000086462:Coronavirus Nucleocapsid Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D016679:Genome, Viral; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D019590:Nucleocapsid Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D045169:Severe Acute Respiratory Syndrome / Q000097:blood / Q000175:diagnosis* / Q000821:virology
D000914:Antibodies, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D007074:Immunoglobulin G; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins