Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein.
The entire coding region of open reading frame 2 (ORF2) of porcine circovirus 2 (PCV2) was linked to the 3'-end of the maltose-binding protein (MBP)-His(8)-tag gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-d-galactoside. MBP-His(8)-ORF2 was purified to homogeneity by immobilized metal affinity chromatography based on the interaction of the polyhistidine-tag with metal ions. Expression could represent 1% of the total protein in Escherichia coli, allowing approximately 1 mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-PCV2 polyclonal antibody and swine sera with PCV2 infection. In addition, rabbit polyclonal antibody raised against purified MBP-His(8)-ORF2 fusion protein reacted with the ORF2 protein in immunoprecipitation. The availability of this fusion protein should permit a thorough study of prevalence of PCV2 infection in large-scale serological studies of field samples.
Liu
Q
Q
Veterinary Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, Saskatchewan, Canada S7N 5E3. liuq@calgary.ca
Willson
P
P
Attoh-Poku
S
S
Babiuk
L A
LA
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D036022:Capsid Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D017925:Circovirus / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006023:Glycoproteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D015183:Restriction Mapping; D013552:Swine
D018528:ATP-Binding Cassette Transporters; D036022:Capsid Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D006023:Glycoproteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli