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Master's Dissertation
DOI
Document
Author
Full name
Aline Sanches Pereira
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2018
Supervisor
Committee
Russo, Elisa Maria de Sousa (President)
Castro, Virgínia Picanço e
Cabral, Hamilton
Fuentes, Andrea Soares da Costa
Title in Portuguese
Expressão de proteínas recombinantes dos vírus Dengue, Zika e Chikungunya para diagnóstico sorológico diferencial das viroses
Keywords in Portuguese
Arboviroses
Diagnóstico
Escherichia coli
Proteína recombinante
Abstract in Portuguese
As arboviroses dengue, zika e chikungunya representam um sério problema de saúde pública no Brasil e no mundo. Elas circulam simultaneamente devido ao transmissor comum, vetores da classe Aedes, e são responsáveis por milhares de casos anuais no Brasil com consequências graves à população, levando à morte muitos indivíduos. Essas doenças possuem sintomas semelhantes, o que dificulta o diagnóstico clínico exato. O diagnóstico específico se mostra necessário para evitar o agravamento das consequências que cada uma pode trazer ao paciente. Os testes diagnósticos sorológicos disponíveis no mercado identificam uma única virose por vez. Os testes discriminatórios são, normalmente, moleculares utilizando PCR em tempo real. Tais métodos são excelentes, mas inadequados para o diagnóstico em massa devido ao custo. Devido a todos os riscos de morte ou de sequelas permanentes que esses vírus impõem aos indivíduos, um diagnóstico discriminatório se faz necessário e melhorias nos conjuntos diagnósticos são desejáveis.Os genes das proteínas ZIKVNS1, DENVNS1, DENVE3 e CHIKVE2 foram clonados com sucesso em diferentes linhagens de Escherichia coli, porém a proteína CHIKVE2 não foi expressa em nenhuma das linhagens. As proteínas ZIKVNS1, DENVNS1 e DENVE3 foram produzidas em bactérias da linhagem Rosetta(DE3) transformada com vetores pET28a recombinantes. Essas três proteínas foram solubilizadas dos corpos de inclusão, purificadas por cromatografia de afinidade ao níquel e identificadas pelas técnicas de SDS-PAGE e Western Blotting. A proteína DENVNS1 foi reconhecida em teste rápido comercial para detecção de antígenos NS1 da dengue o que confere a ela especificidade e grande potencial como antígeno de captura para diagnóstico. Neste trabalho foram produzidas as proteínas recombinantes DENVNS1, ZIKVNS1 e DENVE3 em E. coli. Este é o primeiro passo para produção de um dispositivo diagnóstico rápido, discriminatório, unificado, produzido com tecnologia nacional, reduzindo os gastos com a importação destes produtos e ampliando o alcance diagnóstico.
Title in English
Expression of recombinant protein from Dengue, Zika and Chikungunya viruses to differential serological diagnosis
Keywords in English
Arboviruses
Diagnostic
Escherichia coli
Recombinant protein
Abstract in English
The arboviruses dengue, zika and chikungunya represent a serious public health problem in Brazil and in the world. They circulate simultaneously due to the common vector, Aedes mosquito, and are responsible for thousands of annual reports of infections in Brazil with serious consequences as death of many individuals. These diseases have similar symptoms which impair the exact clinical diagnosis. The specific diagnosis is necessary to avoid aggravating the consequences that each one can bring to the patient. Serological diagnostic tests available on the market identify a single virus at time. Discriminatory tests are molecular assays like real time PCR. Such methods are excellent but inadequate for mass diagnosis due to cost. Because of the risks of death or sequels that these viruses impose on individuals, a discriminatory diagnosis is necessary, and the improvement of tests is desired. The genes of the proteins ZIKVNS1, DENVNS1, DENVE3 and CHIKVE2 were successfully cloned in different strains of Escherichia coli, but the CHIKVE2 protein was not expressed in any of the strains. ZIKVNS1, DENVNS1 and DENVE3 proteins were produced in Rosetta (DE3) bacteria transformed with pET28a recombinant vectors. These three proteins were solubilized from inclusion bodies, purified by nickel affinity chromatography and identified by SDS-PAGE and Western Blotting. The DENVNS1 protein was recognized in a rapid test for detection of dengue NS1 antigens, which gives it specificity and great diagnostic potential as capture antigen. In this work the recombinant proteins DENVNS1, ZIKVNS1 and DENVE3 were successfully expressed in E. coli system and this is the first step in the production of a fast, discriminatory, unified diagnostic device produced with national technology, reducing the costs of importing these products. These rapid tests can broad the diagnostic cover.
 
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Release Date
2021-05-21
Publishing Date
2019-11-14
 
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