J Vet Sci.  2002 Mar;3(1):19-23.

Characterization of the Recombinant Proteins of Porcine Circovirus Type2 Field Isolate Expressed in the Baculovirus System

• Kim Y
• Kim J
• Kang K
• Lyoo YS

Abstract

Porcine circovirus (PCV) type2 was isolated using primary porcine kidney cells from lymph node of piglets with typical PMWS. The presence of the virus was identified by PCR using primers specific to PCV type2. The ORFs 1 and 2 were amplified by PCR using primers corresponding to the target genes of the PCV type 2. Cloned genes were inserted into the baculovirus expression vector and PCV recombinant proteins were expressed using baculovirus expression system. Recombinant protein expression was determined by indirect immunofluorescent assay (IFA) and immunoblotting using polyclonal antiserum to PCV. ORF1 gene expressed two proteins with approximately 17 kDa and 31 kDa proteins in the baculovirus system. Recombinant protein of the ORF2 was similar to that of the native virus except minor bands with different molecular weight were detected. Recombinant protein expressed in the baculovirus system showed at least two glycosylation sites based on the tunicamycin treatment. Recombinant protein of the ORF2 assembled virus-like particle in recombinant virus infected insect cells.

Keyword

PCV type2; recombinant protein; baculovirus system

MeSH Terms

Animals
Baculoviridae/*genetics
Blotting, Western
Circoviridae Infections/*veterinary/*virology
Circovirus/*classification/genetics/isolation & purification/ultrastructure
Cloning, Molecular
Fluorescent Antibody Technique, Indirect
Lymph Nodes/virology
Microscopy, Electron
Open Reading Frames
Palatine Tonsil/virology
Polymerase Chain Reaction/methods/veterinary
Recombinant Proteins/analysis
Swine
Swine Diseases/*virology
Transfection
Tunicamycin/pharmacology
Viral Proteins/*analysis