Avaliação da expressão em células Vero dos candidatos vacinais pCID2EtD2prM e pCID2EtD3prM de DNA recombinante para o vírus Dengue-2

• Ano de defesa: 2009
• Autor(a) principal: Dutra, Nina Rocha
• Orientador(a): Paula, Sérgio Oliveira de
• Banca de defesa: Oliveira, Leandro Licursi de , Fietto, Luciano Gomes
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Biologia Celular e Estrutural
• Departamento: Análises quantitativas e moleculares do Genoma; Biologia das células e dos tecidos
• País: BR
• Palavras-chave em Português: Vírus dengue; Células Vero
• Palavras-chave em Inglês: Dengue virus; Vero cells
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL
• Link de acesso: http://locus.ufv.br/handle/123456789/2352

The dengue viruses have single-stranded, positive polarity RNA genome that encodes proteins in a long open-reading frame (ORF). This genome is about 11 kb-length and responsible by encoding three structural proteins called Capsid (C), membrane (M) and Envelope (E), this last one being a glycoprotein. In addition to these proteins, the viral genome expresses seven other nonstructural proteins called NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5. The dengue viruses belong to the Flaviviridae family, and are the most important human pathogens of this family. They are transmitted by the bite of the Aedes aegypti mosquito, leading to the dengue disease. The manifestations of this disease vary from asymptomatic infections until severe hemorrhagic features which are responsible by the high morbidity and mortality rates of dengue. So far, there is not specific antiviral treatment. Many researchers have been working on the quest for an efficient dengue vaccine, and consequently, various strategies have been brought to view for its production, using simpler methods, such as the viral attenuation, as well as more complex ones, such as the expression of recombinant antigens in expression vectors, like viruses and bacteria. However, the results obtained in the developmente of these vaccines have been frustrating the world scientific community. A new vaccine strategy, the DNA vaccine, is currently under intense investigation. In this context, a vaccinal candidate for the dengue-2 virus (DENV-2) has been constructed utilizing the pCI plasmid vector expressing the truncated E protein from DENV- 2, without the concomitant expression of prM, aiming the size reduction of the insert to be cloned, facilitating the production of a tetravalent vaccine in a single plasmid vector. The testing demonstrated that the truncated E protein was correctly expressed by the plasmid, but in reduced quantity, and that a good specific response existed against the dengue-2 virus generated by the recombinant clone, but only a small percentage of the immunized mice was protected when challenged with DENV-2. These results justify the search for the improvement of this vaccine candidate against DENV-2, with the objective of increasing the expression of the E protein cloned into the vaccine plasmid, thus offering more efficient immunization.This present work aims the improvement of this plasmid by the insertion of the prM/DENV-2 and prM/DENV-3 proteins, to verify if the presence of these proteins in the vaccine construction would increase the expression of E. The new plasmids constructed were called pCID2EtD2prM and pCID2EtD3prM. The results achieved proved the correct cloning and expression of prM/E by transfected Vero cells, and indicated that there was a relative expression increase of 67,02% by pCID2EtD3prM in relation to pCID2EtD2prM, results that are still to be corroborated by absolute tests.