Cloning and expression of chicken anemia virus VP3 protein in Escherichia coli.
Purification of chicken anemia virus (CAV) VP3 protein, expressed in a prokaryotic expression system as histidine-tagged fusion protein is demonstrated in the present study. CAV particle was obtained from infected liver of chicken and DNA was extracted. The VP3 protein gene was amplified from the extracted DNA by polymerase chain reaction (PCR) and cloned. The recombinant expression construct (pTrc-VP3) was identified by PCR and sequencing analysis. Expression of VP3 protein with a molecular mass of approximately 21kDa was confirmed by Western blotting analysis with CAV-specific antibodies. The in vitro expressed VP3 protein was purified to near homogeneity by elution from the gel, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP3 protein was recognized by CAV antibodies in a Western blotting assay. This finding indicates that recombinant VP3 expressed in the pTrcHis2 vector system can be used as antigen to detect anti-CAV antibodies.
Nogueira-Dantas
Eliana Ottati
EO
Department of Pathology, College of Veterinary Medicine, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo, SP, 05508-000, Brazil.
Ferreira
Antonio J Piantino
AJ
Astolfi-Ferreira
Claudete Serrano
CS
Brentano
Liana
L
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D015153:Blotting, Western; D036022:Capsid Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D017727:Chicken anemia virus / Q000737:chemistry / Q000235:genetics*; D002645:Chickens; D018173:Circoviridae Infections / Q000175:diagnosis / Q000662:veterinary; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D011201:Poultry Diseases / Q000175:diagnosis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D000914:Antibodies, Viral; D036022:Capsid Proteins; D004279:DNA, Viral; D011994:Recombinant Proteins; C091717:VP3 protein, Chicken anemia virus