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Mémoire de Maîtrise
DOI
https://doi.org/10.11606/D.87.2011.tde-27092011-151543
Document
Auteur
Nom complet
Renata de Siqueira Mendes
Unité de l'USP
Domain de Connaissance
Date de Soutenance
Editeur
São Paulo, 2011
Directeur
Jury
Nascimento, Ana Lucia Tabet Oller do (Président)
Elias Junior, Waldir Pereira
Soares, Irene da Silva
Titre en portugais
Caracterização de proteínas de membrana de Leptospira interrogans expressas em Escherichia coli.
Mots-clés en portugais
Escherichia coli
Spirochaetales
Genomas
Leptospirose
Proteínas recombinantes
Vacinas
Resumé en portugais
O sequenciamento genômico da L. interrogans sorovar Copenhageni e os avanços das análises bioinformáticas permitiram a identificação de seis novos candidatos vacinais. Esses genes de Leptospira foram submetidos a ensaios de conservação do DNA genômico, RNA mensageiro e proteína nativa correspondente em doze sorovares de Leptospira, nos quais o gene LIC11469 foi o mais conservado. As proteínas recombinantes rLIC11469 e rLIC11030 foram purificadas por cromatografia de afinidade a metal, e submetidas a ensaio de dicroísmo circular. Em ensaios de localização celular pudemos observar a presença das proteínas nativas correspondentes na membrana externa de Leptospira. Ensaios de adesão mostraram a ligação da proteína rLIC11469 à laminina e ao plasminogênio. Ensaios de imunização e desafio demonstraram que a proteína rLIC11030 conferiu proteção contra infecção letal de L. interrogans em hamsters. Ambas as proteínas apresentaram reatividade com anticorpos presentes em soros de pacientes com leptospirose, sugerindo sua expressão durante a infecção.
Titre en anglais
Characterization of membrane proteins of Leptospira interrogans expressed in Escherichia coli.
Mots-clés en anglais
Escherichia coli
Spirochaetales
Genomes
Leptospirosis
Recombinant proteins
Vaccines
Resumé en anglais
The genomic sequencing of the L. interrogans serovar Copenhageni and the advances of bioinformatics analysis allowed the identification of six new vaccine candidates. These genes were subjected to genomic DNA, mRNA and native protein conservation assays in twelve serovars from Leptospira, and the gene LIC11469 was the most conserved among these serovars. The recombinant proteins rLIC11469 and rLIC11030 were purified by metal affinity chromatography, and subjected to circular dichroism. Through cellular localization assays we could observe the presence of the corresponding native proteins on the outer membrane of Leptospira. In adhesion assays, the protein rLIC11469 showed binding to laminin and plasminogen. Immunization and challenge assays showed that the protein rLIC11030 afforded protection against lethal leptospiral inoculation in hamsters. Both proteins showed reactivity against sera of patients diagnosed with leptospirosis, suggesting that these proteins are probably expressed during infection.
 
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Date de Publication
2011-09-29
 
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