Construção e expressão heteróloga de domínio III da proteína de envelope (E) dos vírus dengue -1e - 3 em Pichia pastoris

• Autor(a) principal: Paixão, Vinicius Ferreira da
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2373
• topic: Dengue - Diagnóstico; Dengue - Vacina; Aedes aegypti; Pichia pastoris; Dengue - Diagnosis; Dengue - Vaccine; Aedes aegypti; Pichia pastoris; CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL

Dengue is the most important arboviral disease in the world risking almost half the world's population, classified as a Neglected Tropical Disease. It is imperative the development and improvement of diagnostic methods and vaccines for dengue. However, the development of these are limited by the difficulty in large-scale production of antigens for use in capturing present in serum of infected patients and antibody to immunize guinea pigs. Due to this limiting factor, this work aimed to produce in large antigen quantity and quality of the domain III of the E protein of dengue virus. Therefore, the P. pastoris was used because it is a yeast that has by nature a pattern of post- translational modifications of proteins similar in their patterns of mammals when comparing this genre (Pichia) with other genera of yeasts now used in heterologous expression. Sequences respond to it the domain III of the E protein of DENV- 1 and -3 were inserted DNA construction and pTZ/domIIIDENV1 pTZ/domIIIDENV3 and cloned in E. coli and inserts domIII DENV -1 and -3 were released by digestion with the restriction enzymes EcoRI and NotI and subsequently linked to the yeast expression vector pPICzαA. The resulting constructs were also pPICzαA/domIIIDENV3 pPICzαA/domIIIDENV1 and cloned in E. coli and P. pastoris GS115 they were transformed by electroporation and selected in medium with ZeocinTM. All constructs and transformation was confirmed by PCR. Once obtained recombinant strains was made domIIIDENV1 protein expression and by serological techniques as ELISA IgG and IgM positive patients was confirmed Dengue the great similarity of the expressed heterologous protein and native protein , since these techniques are highly sensitive and specific for depending on the antigen-antibody binding . This work demonstrates the great potential for large-scale production of antigen Domain III of the envelope protein of dengue virus 1 and 3 for construction of diagnostic kits" and improvements in dengue control.