title: [Molecular cloning and expression of the severe acute respiratory syndrome-associated coronavirus nucleocapsid protein and its clinical application].
abstract: 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.
authors.affiliation: Shenzhen Donghu Hospital, Shenzhen, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Lu
authors.forename: Bai-ping
authors.initials: BP
authors.lastname: Zhou
authors.forename: Yu-sen
authors.initials: YS
authors.lastname: Zhou
authors.forename: Xiao-ling
authors.initials: XL
authors.lastname: Jiang
authors.forename: Li-xia
authors.initials: LX
authors.lastname: Wen
authors.forename: Xiao-hua
authors.initials: XH
authors.lastname: Le
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Liu-mei
authors.initials: LM
authors.lastname: Xu
authors.forename: Li-xiong
authors.initials: LX
authors.lastname: Li
mesh_terms: 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
chemical_list: D000914:Antibodies, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D007074:Immunoglobulin G; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins