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Master's Dissertation
DOI
10.11606/D.87.2013.tde-05112013-094703
Document
Author
Full name
Jacqueline Mazzuchelli de Souza
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2013
Supervisor
Committee
Stocco, Rita de Cassia (President)
Kfoury Junior, José Roberto
Mendonça, Ronaldo Zucatelli
Title in Portuguese
Clonagem e expressão do gene E6 do Papilomavírus Bovino do tipo 1.
Keywords in Portuguese
Papovaviridae
Bioinformática
Clonagem animal
Oncologia veterinária
Oncoprotreínas
Abstract in Portuguese
O Papilomavírus Bovino do tipo 1 (BPV-1) causa fibropapilomas em bovinos e sarcóide em equinos, associados à expressão de oncoproteínas virais, principalmente E6 e E7. A purificação de oncoproteínas a partir de sistema recombinante possibilita seu estudo. Os objetivos foram expressar o gene E6 do BPV-1, purificar e analisar in silico a proteína recombinante obtida. O amplificado foi clonado em E. coli e os plasmídeos foram sequenciados, confirmando a matriz correta de leitura. Após indução da expressão, a proteína recombinante foi identificada e purificada. A microscopia eletrônica mostrou a formação de corpúsculos de inclusão. As análises in silico apontaram as mutações das sequências gênica e proteica de E6-1 em relação às depositadas. Foi realizada a predição tridimensional da estrutura da proteína recombinante e identificadas as regiões conservadas, antigênicas e capazes de realizar ligações cátion-p. Logo, a proteína recombinante E6 do BPV-1 foi obtida e purificada com sucesso, possibilitando o início de novos experimentos e estudos mais detalhados.
Title in English
Cloning and expression of Bovine Papillomavirus type 1 E6 gene.
Keywords in English
Papovaviridae
Animal cloning
Bioinformatics
Oncoproteins
Veterinary oncology
Abstract in English
Bovine Papillomavirus type 1 (BPV-1) causes fibropapillomas in cattle and sarcoid in equines, associated with the expression of viral oncoproteins, E6 and E7 particularly. Purification of oncoproteins from recombinant system allows their study. The aim of this study was cloning and expressing BPV-1 E6 gene, purify and analyze in silico recombinant protein. Amplicon was cloned into E. coli and the plasmids were sequenced to confirm the correct reading frame. After induction of expression, the recombinant protein was identified and purified. Electron microscopy showed the inclusion bodies formation. In silico analysis showed mutations in E6-1 gene and protein sequences when there were compared to sequences available in current database. Three-dimensional structure prediction of the recombinant protein was performed and conserved, antigenic and cation-p interaction regions were identified. Therefore, BPV-1 E6 recombinant protein was obtained and successfully purified enabling new experiments and more detailed studies.
 
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Publishing Date
2014-01-16
 
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