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Master's Dissertation
DOI
https://doi.org/10.11606/D.42.2020.tde-17052024-180954
Document
Author
Full name
Jéssica Amaral Martinho
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2020
Supervisor
Committee
Boscardin, Silvia Beatriz (President)
Bordignon, Juliano
Durigon, Danielle Bruna Leal de Oliveira
Spencer, Patrick Jack
 
Title in Portuguese
Produção de proteínas do envelope (E) mutadas no loop de fusão dos quatro sorotipos do vírus da dengue e do vírus Zika.
Keywords in Portuguese
Células S2
Dengue
Imunidade humoral
Proteína do envelope
Zika
Abstract in Portuguese
A reatividade cruzada presente nos vírus da família Flaviviridae é um dos fatores que levam a resultados falsos positivos no diagnóstico dessas infecções. Desta forma, o uso de reagentes eficientes que permitam o diagnóstico preciso é essencial. Este trabalho tem como objetivo a produção de proteínas do envelope (E) dos quatro sorotipos do vírus da dengue e do vírus Zika, contendo quatro mutações pontuais próximas e na região do loop de fusão, com o objetivo de reduzir a reatividade cruzada esses flavivírus. Para tal, utilizamos sequências consensos das proteínas E dos quatro sorotipos do vírus da dengue e do vírus Zika contendo mutações nos seguintes aminoácidos: T76R, Q77E, W101R e L107R conforme descrito previamente. As sequências foram submetidas a otimização de códons e clonadas em vetor de expressão para células de Drosophila melanogaster. Após a confirmação da expressão das proteínas na forma solúvel por transfecção transiente, foram estabelecidas linhagens estáveis. Neste trabalho foi possível obter linhagens estáveis expressando três das quatro proteínas E mutadas do vírus da dengue (DENV2, DENV3 e DENV4) e a proteína E do vírus Zika. A caracterização das proteínas obtidas foi realizada por ELISA utilizando-se anticorpos monoclonais humanos previamente gerados em nosso laboratório (MT600-A3, MT479-C4,MT479-D2 e MT479-F11) e anticorpos monoclonais murinos (4G2, 1A10 e anti-HisTag) contra a proteína E, além das proteínas E do DENV2 e DENV3 selvagenscomo controles. Os anticorpos monoclonais humanos foram capazes de reconhecermelhor as proteínas selvagens. Para as proteínas mutadas, os anticorpos MT479-C4 e MT479-D2 foram capazes de reconhecer a proteína E mutada do DENV4. O anticorpo MT479-F11 não foi capaz de reconhecer as proteínas mutadas. Já o MT600-A3 não teve um reconhecimento significativo para qualquer das roteínasmutadas. Quando analisamos os anticorpos murinos, observamos que o 1A10 e o4G2 apresentaram elevado reconhecimento para as proteínas E selvagens emcomparação com as proteínas E mutadas, que foram mais fracamente reconhecidas. A obtenção destas proteínas abre perspectivas para sua testagem em testes sorológicos que visem melhorar a especificidade e separar de forma segura infecções pelos vírus dengue e Zika.
 
Title in English
Production of envelope (E) proteins mutated in the fusion loop derived from the four dengue virus serotypes and from zika virus.
Keywords in English
Dengue
Envelope protein
Humoral immunity
S2 cells
Zika
Abstract in English
The cross-reactivity present in the viruses of the family Flaviviridae is one of the factors that leads to false positive results in the diagnosis of these infections. Therefore, the use of efficient reagents that allow an accurate diagnosis is essential. This work aimed to produce envelope (E) proteins from the four dengue virus serotypes and from zika virus, containing four point mutations close to and in the fusion loop region, with the objective of reducing the cross-reactivity of these flaviviruses. For this, we used consensus sequences of E proteins of the four serotypes of dengue virus and of zika virus containing mutations in the following amino acids: T76R, Q77E, W101R and L107R, as previously described. The sequences were subjected to codon optimization and cloned into an expression vector for Drosophila melanogaster cells. After confirmation of protein expression in the soluble form by transient transfection, stable cell lines were established. In this work it was possible to obtain stable cell lines expressing three of the four mutated E proteins of the dengue virus (DENV2, DENV3 and DENV4) and the protein E of zika virus. The characterization of the obtained proteins was performed by ELISA using human monoclonal antibodies previously generated in our laboratory (MT600-A3, MT479-C4, MT479-D2 and MT479-F11), and murine monoclonal antibodies (4G2, 1A10 and anti-HisTag) against protein E, in addition to wild-type DENV2 and DENV3 E proteins as controls. Human monoclonal antibodies were able to better recognize wild-type proteins. For the mutated proteins, the MT479-C4 and MT479-D2 antibodies were able to recognize the DENV4 mutated E protein. MT479-F11 antibody was unable to recognize mutated proteins. MT600-A3 did not presente significant recognition for any of the mutated proteins. When we analyzed the murine antibodies, we found that 1A10 and 4G2 showed high recognition for wild type E proteins compared to mutated E proteins, which were more poorly recognized. Obtaining these proteins opens up perspectives for their testing in serological tests that aim to improve specificity and safely separate infections by dengue and zika viruses.
 
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Release Date
2026-05-17
Publishing Date
2024-06-11
 
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