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Doctoral Thesis
DOI
10.11606/T.87.2009.tde-17042009-154849
Document
Author
Full name
Fernando Moreira Simabuco
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Ventura, Armando Morais (President)
Ferreira, Rita de Cassia Cafe
Ho, Paulo Lee
Marques, Marilis do Valle
Zanchin, Nilson Ivo Tonin
Title in Portuguese
Expressão das proteínas N e P do vírus respiratório sincicial humano: estudos funcionais e de imunização.
Keywords in Portuguese
Biologia molecular
Bioquímica
Expressão gênica
Proteínas
Vacinas
Vírus de RNA
Abstract in Portuguese
O Vírus Respiratório Sincicial Humano é um vírus envelopado de RNA negativo e considerado o patógeno mais importante do trato respiratório de bebês e crianças. As proteínas virais N e P foram expressas em bactérias, purificadas e usadas para a produção de anticorpos policlonais em camundongos. Estudos in silico permitiram a predição de regiões desordenadas da proteína P, as quais foram identificadas por espectrometria de massa como regiões hiper sensíveis a proteases. A otimização dos genes N e P permitiu uma forte e eficiente expressão das proteínas N e P em células humanas. Vacinas de DNA contendo os genes otimizados foram testadas em camundongos e geraram forte resposta imune humoral. As proteínas N e P expressas em células humanas foram imunoprecipitadas e analisadas quanto a interações com proteínas celulares, identificadas por espectrometria de massa. A proteína P mostrou ser capaz de interagir com a HSP70. Por fim, um sistema Minigenoma alternativo, usando o promotor da RNA polimerase II, foi construído para o HRSV mas pouca ou nula atividade foi detectada.
Title in English
Expression of human respiratory syncytial virus N and P proteins: functional and immunization studies.
Keywords in English
Biochemistry
Genetic expression
Molecular biology
Proteins
RNA viruses
Vaccines
Abstract in English
The Human Respiratory Syncytial Virus is a single stranded negative RNA enveloped virus and it is considered the most important pathogen of the respiratory tract of infants and neonates. The viral N and P proteins were expressed in bacteria, purified and used for the production of polyclonal antibodies in mice. In silico studies allowed the prediction of intrinsically disordered domains for P protein, which were identified by mass spectrometry as protease hyper sensible regions. The optimization of N and P genes allowed a robust expression of the N and P proteins in human cells. DNA vaccines containing the optimized genes were tested in mice and generated strong humoral imune response. The N and P proteins expressed in human cells were immunoprecipitated and their interactions with cellular proteins were identified by mass spectrometry. The P protein was able to interact with the HSP70 protein. Finally, an alternative Minigenome system, using an RNA polymerase II promoter, was developed for HRSV but low or no activity was detected.
 
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Publishing Date
2009-04-27
 
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