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Doctoral Thesis
DOI
10.11606/T.87.2009.tde-11022010-094355
Document
Author
Full name
Renato Mancini Astray
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2009
Supervisor
Committee
Pereira, Carlos Augusto (President)
Kedinger, Claude
Pattus, Franc
Schenberg, Ana Clara Guerrini
Ventura, Armando Morais
Wagner, Renaud
Title in Portuguese
Expressão da glicoproteína recombinante do vírus rábico em sistemas Drosophila melanogaster (S2) e Semliki Forest Virus (SFV).
Keywords in Portuguese
Células S2
ELISA
Glicoproteína do vírus rábico
qRT-PCR
RVGPmRNA
Semliki Forest virus
Abstract in Portuguese
A glicoproteína do vírus rábico (RVGP) é o antígeno capaz de induzir a formação de anticorpos neutralizantes e a resposta imune protetora contra a infecção pelo vírus rábico. Estudamos as cinéticas de expressão da RVGP e de seu RNA mensageiro (RVGPmRNA) em dois sistemas distintos de expressão recombinante: células de Drosophila melanogaster (S2) e células BHK-21 infectadas com vírus Semliki Forest Virus (SFV). Para isso, fizemos um trabalho de padronização do tratamento de amostras de cultivos celulares, adequando-as a um teste de ELISA para dosagem da RVGP e estabelecemos um método de RT-PCR quantitativa (qRT-PCR) para a dosagem do RVGPmRNA. Desenvolvemos também um novo método de titulação de partículas SFV por qRT-PCR, aplicável a praticamente qualquer construção de SFV recombinante. Em ensaios preliminares, as preparações de RVGP recombinante utilizadas para a imunização de camundongos foram capazes de induzir a formação de anticorpos neutralizantes e de conferir um bom grau de proteção ao teste de desafio intracerebral com vírus rábico.
Title in English
Rabies virus glycoprotein expression in Drosophila melanogaster (S2) and Semliki Forest Virus (SFV) systems.
Keywords in English
ELISA
qRT-PCR
Rabies virus glycoprotein
RVGPmRNA
S2 cells
Semliki Forest virus
Abstract in English
The rabies virus glycoprotein is the major antigen able to induce a neutralizing antibody response and survival after challenge against rabies virus infection. We have studied the kinetic expression of RVGP and its messenger RNA (RVGPmRNA) in two different recombinant expression systems: stably transfected Drosophila melanogaster cells (rS2) and BHK-21 cells infected with Semliki Forest Virus carrying RVGP genetic information (SFV-RVGP). We have done a work of standardization of the cell culture samples treatment prior to RVGP quantification by ELISA, and we have developed and standardized a quantitative RT-PCR (qRT-PCR) to quantify the RVGPmRNA. We have also developed a new method of SFV particles titration by qRT-PCR, which is applicable to other constructions of recombinant SFV. We utilized the RVGP expressed by rS2 and SFV-RVGP systems on preliminary in vivo assays. The RVGP samples used to mice immunization were able to induce neutralizing antibodies and to lead to a nice level of protection against the intracerabral rabies virus challenge.
 
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Publishing Date
2010-02-18
 
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