Circovírus suino tipo 2 (PCV2): caracterização molecular e construção de vetores para expressão da proteína do capsídeo

• Autor(a) principal: Silva Júnior, Abelardo
• Data de Publicação: 2005
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2395
• topic: Circovírus suino; Vacinas recombinantes; Análise molecular; Porcine circovirus; Recombinant vaccines; Molecular analysis; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

PCV presents a single-stranded circular DNA genome of approximately 1760 bp, with two main open reading frames (ORFs): ORF1 that encodes viral replicase and ORF2 that encodes viral capsid protein. The aim of this work was to perform a molecular characterization of a PCV2 isolate and produce an expression vector containing the sequence of viral capsid protein. PCV2 DNA extracted from infected SK6 cells, was amplified by PCR and then confirmed by sequencing. After confirmation of PCV2 isolation, all viral genome was amplified by PCR and inserted in a cloning vector, and the recombinant clone was sequenced. To ORF2 subcloning, PCR assays that amplified the genomic segment of capsid protein were performed and the PCR product was inserted in an expression vector. This plasmidial DNA was transfected in SK6 cells free of PCV. The expression of the viral protein was confirmed by indirect imunofluorescence methods. ORF1 complete nucleotide sequence analysis and ORF2 partial nucleotide sequence analysis revealed respectively, 99.27% and 97.99% of identity with sequences retrieved from GenBank. Until now there is no available vaccinal method to control swine circovirosis. The results obtained in this work will be of great importance to the beginning of vaccination studies in animal models.