Construção de um sistema de expressão da proteína não estrutural (NS1) do vírus dengue-2 em Nicotiana tabacum "Havana" e análise da expressão do transgene

• Ano de defesa: 2009
• Autor(a) principal: Amaro, Marilane de Oliveira Fani
• Orientador(a): Paula, Sérgio Oliveira de
• Banca de defesa: Fietto, Luciano Gomes , Fietto, Juliana Lopes Rangel
• 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: Dengue; Nicotiana havana; Agrobacterium tumefaciens; Expressão heteróloga
• Palavras-chave em Inglês: Dengue; Nicotiana havana; Agrobacterium tumefaciens; Heterologous expression
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL
• Link de acesso: http://locus.ufv.br/handle/123456789/2322

The dengue is the most important arbovirus disease in the world, being observed in the last twenty years a significant increase in the epidemic activity, expansion of the geographical distribution, continuous transmission of several sorotypes and emergency of the Hemorrhagic Fever (DHF) in areas where the disease was not prevalent. In spite of the methodological processes of the dengue's diagnosis are in deep development and improvement, a difficulty in the accomplishment of diagnostic tests for dengue lives in the difficult production of antigens in great scale to be used in the capture of the present antibody in the infected patients serum. Thus, our aim in this work is to obtain antigen in great amount and quality using a system of heterologous expression of proteins in plants. To achieve that, total RNA of the culture supernatant of infected cells was extracted and the cDNA was made. The gene of the non structural protein (NS1) of the sorotype dengue-2 was obtained by Polimerase Chain Reactions (PCR), separate by eletroforesis and purified. The gene was, then, cloned in pGEM-T and pCAMBIA vectors. Escherichia coli DH5α were transformed, selected in selective medium and stored at -80 °C. The vector pCAMBIA was previously cleaved with the same enzymes and the gene of the NS1 protein was binded to it. New transformation of E. coli DH5α was accomplished and the colonies were, then, analyzed for the presence of NS1 gene by PCR. The transformed recombinant colonies were selected and stored. The recombinant vector, pCAMBIA 3301/NS1, was used for transformation in Agrobacterium tumefaciens and, later, in Nicotiana tabacum for expression of the non-structural protein NS1. DNA and RNA extraction from the tobacco plants was accomplished. The integration of the NS1 protein gene in the plant genome and its transcription were confirmed by PCR. Subsequent analyses will be accomplished to verify its effective expression, and it will be further purified and concentrated.