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Doctoral Thesis
Full name
Sandra Fernanda Suárez Patiño
Knowledge Area
Date of Defense
São Paulo, 2016
Jorge, Soraia Attie Calil (President)
Azzoni, Adriano Rodrigues
Lemos, Marcos Alexandre Nobre
Mendonça, Ronaldo Zucatelli
Soares, Carlos Roberto Jorge
Title in Portuguese
Produção de proteínas recombinantes em células BHK-21 cultivadas em meio livre de soro fetal bovino.
Keywords in Portuguese
Semliki Forest Virus
Células BHK-21
Glicoproteína do vírus da raiva
Meio livre de soro
Proteína não estrutural 3 do vírus da hepatite C
Proteína verde fluorescente
Soro fetal bovino-SFB
Abstract in Portuguese
Células eucariotas usadas como plataforma de expressão de proteínas recombinantes são geralmente cultivadas com soro fetal bovino (SFB), porém, abordagens biotecnológicas atuais sobre cultura de células devem evitar o uso deste suplemento, devido a problemas de custo, variações entre os lotes e risco de contaminação. Assim, nosso objetivo foi expressar as proteínas recombinantes: GFP (proteína verde fluorescente), NS3 (proteína não estrutural 3 do vírus da hepatite C) e RVGP (glicoproteína do vírus da raiva) em células BHK-21 adaptadas em meios livres de soro fetal bovino (SFM) usando o sistema de expressão baseado no Semliki Forest Virus (SFV). Os resultados do presente trabalho mostraram que células adaptadas em SFM cresceram de forma eficiente, produziram mais partículas virais recombinantes de SFV do que células suplementadas com soro, sendo que estas partículas virais podem ser usadas diretamente para imunização, pois garantiram uma amplificação e expressão eficiente das diferentes proteínas dentro da célula hospedeira.
Title in English
Production of recombinant proteins in BHK-21 cells cultured in serum free media.
Keywords in English
BHK-21 cells
Fetal bovine serum
Green fluorescent protein
Hepatitis C virus non-structural protein 3
Rabies virus glycoprotein
Semliki Forest Virus
Serum-free medium
Abstract in English
Eukaryotic cells are cultured with serum, however current biotechnological approaches of cell culture need to avoid using of this supplement, due to the high costs, lot-to-lot variation and risk of contamination. Thus, our aim was to express the recombinant protein: GFP (green fluorescent protein); NS3 (Hepatitis C virus non-structural protein 3) and RVGP (rabies virus glycoprotein) in BHK-21 cells cultured in serum free culture based on Semliki Forest Virus system. The results of this work showed that cells cultured in serum-free media (SFM) were grown efficiently, they were produce more recombinant viral particles when compared with cells supplemented with SFB. These viral particles can be used directly for immunization, since generated amplification and expression efficient of different proteins within the host cell.
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