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Doctoral Thesis
DOI
https://doi.org/10.11606/T.87.2009.tde-28042009-123838
Document
Author
Full name
Rodrigo Esaki Tamura
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Ventura, Armando Morais (President)
Harsi, Charlotte Marianna
Mendonça, Ronaldo Zucatelli
Monteiro, Hugo Pequeno
Strauss, Bryan Eric
Title in Portuguese
Estudo dos domínios funcionais da  proteína de matriz do vírus respiratório sincicial humano.
Keywords in Portuguese
Citoesqueleto
Membrana plasmática
Núcleo celular
Proteínas recombinantes
Vacinas
Vírus de RNA
Abstract in Portuguese
A proteína de matriz do Virus Respiratório Sincicial humano foi o foco deste trabalho. Verificamos que o gene de matriz possui sítios internos de poliadenilação, sinais de instabilidade de RNA, baixo índice de adaptação de codons (CAI) e conteúdo GC, que podem impedir a expressão gênica vitro. Quando clonado sob controle do promotor de CMVie, o gene selvagem não apresenta expressão detectável, enquanto um gene sintético com a sequência do gene de matriz otimizada apresenta altos níveis de expressão em células transfectadas. Esse alto nível de expressão permitiu a confirmação da presença da proteína M no núcleo no início de sua expressão, por análise em microscopio confocal de varredura a laser, além de sua associação a membranas em regiões conhecidas como lipid rafts. Também foi observado que a proteína M é capaz de associar à proteína tropomiosina. Ainda foram analisados os possíveis domínios funcionais através de expressão de variantes da proteína M com deleções de trechos da proteína. Finalmente foi analisada a capacidade de indução de resposta imune.
Title in English
Study of the human respiratory syncytial virus matrix protein functional domains.
Keywords in English
Cell nucleus
Citoeskeleton
Plasma membrane
Recombinant proteins
RNA virus
Vaccines
Abstract in English
The Human Respiratory Syncytial Vírus was the focus of this work. We found that matrix gene has internal polyadenilation sites, RNA instability motifs, low codon adaptation index (CAI) and GC content, that may impair its expression in vitro. When cloned under control of the ieCMV promoter, the wild-type M gene expression was not detectable, whereas a synthetic optimized matrix gene was highly expressed in transfected cells. This high level of expression made possible to follow M nuclear localization in the beginning of its expression by confocal laser scanning microscopy, and its association with membranes in regions known as lipid rafts. It has also been found that the matrix protein associates with tropomyosin. It was further analyzed the possible functional through expression of deviations of the M protein that lack portions of the protein. Finally it was analyzed its capacity to induce an immune response.
 
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Publishing Date
2009-05-05
 
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