[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.

CONCLUSION:

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: Jian Lu, Bai-ping Zhou, Yu-sen Zhou, Xiao-ling Jiang, Li-xia Wen, Xiao-hua Le, Bing Li, Liu-mei Xu, Li-xiong Li
• Journal: Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology (Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi) Vol. 19 Issue 1 Pg. 64-7 (Mar 2005) ISSN: 1003-9279 [Print] China
• PMID: 16201478 (Publication Type: Journal Article)

Chemical References

• Antibodies, Viral
• Coronavirus Nucleocapsid Proteins
• Immunoglobulin G
• Nucleocapsid Proteins
• RNA, Viral
• Recombinant Proteins

Topics

• Antibodies, Viral (blood)
• Blotting, Western
• Cloning, Molecular
• Coronavirus Nucleocapsid Proteins
• Enzyme-Linked Immunosorbent Assay
• Gene Expression
• Genome, Viral
• Humans
• Immunoglobulin G (blood)
• Nucleocapsid Proteins (genetics, immunology, metabolism)
• RNA, Viral (genetics)
• Recombinant Proteins (isolation & purification, metabolism)
• Reverse Transcriptase Polymerase Chain Reaction
• Severe acute respiratory syndrome-related coronavirus (genetics, immunology, isolation & purification, metabolism)
• Severe Acute Respiratory Syndrome (blood, diagnosis, virology)