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Doctoral Thesis
DOI
10.11606/T.10.2007.tde-27062007-134340
Document
Author
Full name
Eliana Ottati Nogueira Dantas
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2007
Supervisor
Committee
Ferreira, Antônio José Piantino (President)
Brentano, Liana
Durigon, Edison Luiz
Jerez, José Antonio
Soares, Rodrigo Martins
Title in Portuguese
Clonagem e expressão da proteína VP3 do vírus da anemia infecciosa das galinhas (CAV)
Keywords in Portuguese
CAV
Clonagem
Proteína
pTrcHis2
VP3
Abstract in Portuguese
A purificação da proteína VP3 do vírus da anemia das galinhas (CAV), expressada em um sistema de expressão procariótico como uma proteína de fusão com cauda de histidina, está demonstrada neste estudo. Extraiu-se o DNA da partícula do CAV, obtida de fígado de galinha infectado. Amplificou-se o gene da proteína VP3 a partir do DNA extraído pela reação da polimerase em cadeia (PCR), para subseqüentes clonagem e expressão protéica. O produto recombinante da expressão (pTrc-VP3) foi identificado por PCR e sequenciamento. A técnica de Western blotting determinou a expressão da proteína de fusão VP3 com cauda de histidina de peso molecular de aproximadamente 21 KDa. Através da eluição do gel, obteve-se a purificação da proteína VP3 expressada com homogeneidade julgada pelo gel de poliacrilamida e dodecil sulfato de sódio. A proteína VP3 purificada foi reconhecida por anticorpos do CAV no método Western blotting. Este resultado indica que a proteína VP3 recombinante expressada no sistema do vetor pTrcHis2 pode ser utilizada como antígeno para detectar anticorpos contra o CAV.
Title in English
Cloning and expression of chicken anemia virus VP3 protein
Keywords in English
CAV
Cloning
Protein
pTrcHis2
VP3
Abstract in English
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 21 KDa 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.
 
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Publishing Date
2007-06-28
 
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