Expressão dos genes sintéticos prM e E do vírus dengue-3 em Pichia pastoris: avaliação do uso em diagnóstico da dengue e análise do potencial imunogênico das proteínas

• Autor(a) principal: Teixeira, Michelle Dias de Oliveira
• Data de Publicação: 2014
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/276
• topic: Dengue - Vacina; Diagnóstico; Pichia pastoris; Dengue - Vascina; diagnosis; Pichia pastoris; CNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::NUTRICAO E ALIMENTACAO ANIMAL::AVALIACAO DE ALIMENTOS PARA ANIMAIS

Dengue is a major arbovirus affecting humans today. With the growing number of cases, it is essential to have large-scale production of antigens for the development of diagnostic kits for the rapid detection of patients infected by the virus and consequent proper medical intervention for them. Furthermore, there is no effective vaccine available against this disease that affects much of the world's population. In this work we express the prM and E proteins of dengue-3 virus in yeast Pichia pastoris KM71H. The proteins were produced in soluble form in the supernatant of the culture in the form of "virus-like particles" (VLPs). The supernatant of yeast culture was subjected to protein purification by affinity chromatography using a nickel column, but without success. Thus, the supernatant was subjected to precipitation with increasing concentrations of ammonium sulfate. After confirmation by Western Blot, the fraction of 20% of ammonium sulfate was used as antigen in an indirect ELISA test, using patients serum providing a sensitivity of 73.91% and specificity of 82.61%.The fraction from the precipitation with 80% proved more satisfactory, providing a test with 82.61% of sensitivity and 89.25% specificity for the detection of specific IgM antibodies. Further, the fraction of 80% was used for the detection of specific IgG antibodies against the virus, providing 76.09% of sensivity and 84.78% of specificity. The fractions of 20% and 80% were also evaluated for their ability to generate immune responses against dengue-3 virus in mice. An induction of immune response Th2 was observed, checked by increased production of IL-4 and IL-10 when the cells from the spleens of immunized animals were stimulated with dengue-3 virus. Also there as a induction of specific antibodies in these animals, as shown by indirect immunofluorescence using the sera of mice immunized. Moreover, the neutralizing ability of such antibodies was determined with a title of 512. Also an increase was detected in animals immunized with the recombinant protein in the population of activated TCD4 cells and central memory TCD4 cells. Thus, the methodology proposed here showed promise for obtaining antigens of dengue viruses in search for an effective vaccine candidate and creating quick and inexpensive diagnostic tests, which is of great value since large portions of the areas affected by this disease are economically neglected.