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Doctoral Thesis
DOI
10.11606/T.42.2013.tde-04062014-141509
Document
Author
Full name
Rafael Ciro Marques Cavalcante
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2013
Supervisor
Committee
Ferreira, Luis Carlos de Souza (President)
Martins, Elizabeth Angelica Leme
Miyaji, Eliane Namie
Schenberg, Ana Clara Guerrini
Winter, Carlos Eduardo
Title in Portuguese
Desenvolvimento de sistemas de expressão heteróloga para Bacillus subtilis.
Keywords in Portuguese
Listeria
Antígenos de bactérias
Plasmídeos
Proteínas recombinantes
Vacinas
Abstract in Portuguese
Bacillus subtilis é uma alternativa ao emprego de Escherichia coli para a produção de proteínas recombinantes. O principal entrave à utilização de B.subtilis para esse fim é a baixa disponibilidade de sistemas de expressão. Nesse trabalho, testamos diferentes plasmídeos e promotores com o objetivo de desenvolver sistemas de expressão heteróloga eficientes. Ao fim do trabalho, propomos dois novos sistemas de expressão baseados do arcabouço do plasmídeo pMTL500E e nos promotores dos genes cdd e gsiB, ambos de B.subtilis. Os dois plasmídeos construídos apresentam expressão constitutiva e demonstraram desempenho superior no tocante à produção de proteínas heterólogas quando comparados ao único sistema comercialmente disponível, conhecido como pHT01. Em uma segunda parte do trabalho, propomos a utilização de Listeria innocua como veículo de entrega para antígenos vacinais. Por meio de ensaios ex-vivo e in vivo, demonstramos que essa bactéria possui potencial promissor para aplicações vacinais, inclusive quando comparada ao bem estabelecido B.subtilis.
Title in English
Development of heterologous expression system for Bacillus subtilis.
Keywords in English
Listeria
Bacterial antigens
Plasmids
Recombinant proteins
Vaccines
Abstract in English
Bacillus subtilis is an alternative to the use of Escherichia coli for the production of recombinant proteins. The main bottleneck to the use of B. subtilis for this purpose is the low availability of expression systems. In this study, we evaluated different plasmids and promoters with the aim of developing efficient heterologous expression systems. At the end of this work, we propose two new expression systems based on plasmid pMTL500E and the promoters from cdd and gsiB genes, both of them from B.subtilis. The two plasmids constructed exhibit constitutive expression and demonstrated superior performance regarding the production of heterologous proteins compared to the unique commercially available system, which is known as pHT01. In a second part of the work, we propose the use of Listeria innocua as a delivery vehicle for vaccine antigens. After ex-vivo and in-vivo experiments, we demonstrated that this bacterium has a promising potential for vaccine applications, even when compared to well established B.subtilis.
 
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Publishing Date
2014-06-05
 
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