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Doctoral Thesis
DOI
10.11606/T.87.2005.tde-27102005-163558
Document
Author
Full name
Marcelo da Silva
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2005
Supervisor
Committee
Leite, Luciana Cezar de Cerqueira (President)
Lebrun, Ivo
Pessoa Junior, Adalberto
Ramos, Celso Raul Romero
Teixeira, Lucia Martins
Title in Portuguese
Clonagem, expressão e purificação das proteínas de superfície, PsaA e fragmentos de PspA de Streptococcus pneumoniae
Keywords in Portuguese
Clonagem
Pneumococos
Pneumonia
Proteínas recombinantes
Purificação de PsaA e PspA
Steptococcus
Vacinas
Abstract in Portuguese
Streptococcus pneumoniae é o principal causador da pneumonia bacteriana. As vacinas atualmente disponíveis contêm polissacarídeo capsular conjugado ou não com proteínas carreadoras. No entanto, elas apresentam elevado custo ou proteção reduzida nos grupos de risco (crianças abaixo de 5 anos de idade e idosos). Proteínas de superfície de S. pneumoniae, como a PsaA e PspA, são consideradas fortes candidatas vacinais. Com o objetivo de se desenvolver uma vacina de ampla cobertura e baixo custo contra pneumococos, os genes psaA e pspA foram clonados em vetores de expressão em E. coli, pAE e pET e as proteínas expressas foram purificadas por cromatografias de afinidade e de troca aniônica. O rendimento de proteína recombinante obtido com a construção baseada em pET foi 3 vezes maior que o obtido com pAE. Condições de cultivo foram estabelecidas utilizando meio definido com indução por IPTG e/ou por lactose. As cepas recombinantes estão adequadas para serem usadas em estudos para escalonamento da produção em biorreatores.
Title in English
Cloning, expression and purification of proteins of surface, PsaA and fragments of PspA from Streptococcus pneumoniae
Keywords in English
Cloning
Genic expression
pneumococcus
Purification of PsaA and PspA
Recombinant Proteins
Streptococcus
Vaccines
Abstract in English
Streptococcus pneumoniae is the main causative agent of bacterial pneumonia. The current vaccines available contain capsular polysaccharide conjugated or not with carrier proteins. However these are either too expensive or do not protect the high-risk groups. Surface proteins of S. pneumoniae, such as PsaA and PspA, are considered strong vaccine candidates. With the aim of developing a broad-coverage and low-cost vaccine against pneumococcus, the psaA and pspA genes were cloned in E. coli expression vectors, pAE and pET and the expressed proteins were purified through affinity and anion exchange chromatography. The yield of the recombinant protein obtained with the construction based in pET was 3-fold higher than that obtained with pAE. Culture conditions were established using defined media with IPTG and/or lactose induction. The recombinant strains are now ready to undergo studies for scale-up of production in bioreactors.
 
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2005_005.pdf (1.65 Mbytes)
Publishing Date
2007-09-05
 
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