Characterization of the recombinant proteins of porcine circovirus type2 field isolate expressed in the baculovirus system.
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.
Kim
Yuna
Y
Immnunopathology Lab College of Veterinary Medicine Konkuk University Seoul, 143-701, Korea.
Kim
Jinhyun
J
Kang
Kyoungsoo
K
Lyoo
Young S
YS
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D018173:Circoviridae Infections / Q000662:veterinary* / Q000821:virology*; D017925:Circovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D003001:Cloning, Molecular; D019084:Fluorescent Antibody Technique, Indirect; D008198:Lymph Nodes / Q000821:virology; D008854:Microscopy, Electron; D016366:Open Reading Frames; D014066:Palatine Tonsil / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D011994:Recombinant Proteins / Q000032:analysis; D013552:Swine; D013553:Swine Diseases / Q000821:virology*; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D014764:Viral Proteins / Q000032:analysis*
D011994:Recombinant Proteins; D014764:Viral Proteins; D014415:Tunicamycin