title: Expression and purification of turkey coronavirus nucleocapsid protein in Escherichia coli.
abstract: Purification of turkey coronavirus (TCoV) nucleocapsid (N) protein, expressed in a prokaryotic expression system as histidine-tagged fusion protein is demonstrated in the present study. Turkey coronavirus was partially purified from infected intestine of turkey embryo by sucrose gradient ultracentrifugation and RNA was extracted. The N protein gene was amplified from the extracted RNA by reverse transcription-polymerase chain reaction and cloned. The recombinant expression construct (pTri-N) was identified by polymerase chain reaction and sequencing analysis. Expression of histidine-tagged fusion N protein with a molecular mass of 57 kd was determined by Western blotting analysis. By chromatography on nickel-agarose column, the expressed N protein was purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The protein recovery could be 2.5 mg from 100 ml of bacterial culture. The purified N protein was recognized by antibody to TCoV in Western blotting assay. The capability of the recombinant N protein to differentiate positive serum of turkey infected with TCoV from normal turkey serum was evident in enzyme-linked immunosorbent assays (ELISA). These results indicated that the expressed N protein is a superior source of TCoV antigen for development of antibody-capture ELISA for detection of antibodies to TCoV.
authors.affiliation: Department of Veterinary Pathobiology and Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN 47907-1175, USA.
authors.forename: C C
authors.initials: CC
authors.lastname: Loa
authors.forename: T L
authors.initials: TL
authors.lastname: Lin
authors.forename: C C
authors.initials: CC
authors.lastname: Wu
authors.forename: T A
authors.initials: TA
authors.lastname: Bryan
authors.forename: T
authors.initials: T
authors.lastname: Hooper
authors.forename: D
authors.initials: D
authors.lastname: Schrader
mesh_terms: D000818:Animals; D001483:Base Sequence; D004752:Coronavirus, Turkey / Q000254:growth & development* / Q000302:isolation & purification*; D017931:DNA Primers; D004625:Embryo, Nonmammalian / Q000821:virology; D004753:Enteritis, Transmissible, of Turkeys / Q000196:embryology / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000821:virology*; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification; D014422:Turkeys / Q000196:embryology
chemical_list: D017931:DNA Primers; D019590:Nucleocapsid Proteins; D012367:RNA, Viral