TI:Molecular cloning and expression of the severe acute respiratory syndrome2associated coronavirus nucleocapsid protein and its clinical application 　
AU:LU Jian , ZHOU Baiping , ZHOU Yusen , JIANG Xiaoling ,WEN Lixia ,LE Xiaohua , LI Bing , XU Liumei , LI Lixiong
AD:Shenzhen Donghu Hospital , Shenzhen 518020 ,China
【Abstract】　Objective 　To clone and express nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)2associated coronavirus , and to evaluate its antigenicity and application value in the development of serological diagnostic test for SARS1 Methods 　SARS2associated coronavirus N protein gene was amplified from its genomic RNA by reverse transcript nested polymerase chain reaction (RT2nested2PCR) and cloned into pBADP Thio2TOPO prokaryotic expression vector1 The recombinant N fusion protein was expressed and purified ,and its antigenicity and specificity was analyzed by Western Blot ,to establish the recombinant N protein2based ELISA for detection of IgG antibodies to SARS2associated coronavirus ,and SARS2associated coronavirus lysates2based ELISA was compared parallelly1 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 lysates1 Conclusion 　The recombinant SARS2associated coronavirus N protein possessed better antigenicity and specificity and could be employed to establish a new , sensitive ,and specific ELISA for SARS diagnosis1
【Key words】　Severe acute respiratory syndrome ; 　Coronaviridae ; 　Nucleocapsid proteins ; 　Gene expression ; 　Enzyme2linked immunosorbant assay