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

Detalhes bibliográficos
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
Resumo: 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.
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spelling Teixeira, Michelle Dias de Oliveirahttp://lattes.cnpq.br/0612903958610514Cardoso, Silvia Almeidahttp://lattes.cnpq.br/6041368188542057Paula, Sérgio Oliveira dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4767540P4Damaso, Clarissahttp://lattes.cnpq.br/3900177177421722Eller, Monique Renonhttp://lattes.cnpq.br/4821785523906789Siqueira-batista, Rodrigohttp://lattes.cnpq.br/7992589011048146Gomes, Andréia Patríciahttp://lattes.cnpq.br/27229087047815242015-03-26T12:10:47Z2015-01-162015-03-26T12:10:47Z2014-09-09TEIXEIRA, Michelle Dias de Oliveira. Expression of synthetic genes prM and E of dengue-3 virus in Pichia pastoris: evaluation of use in dengue diagnosis and analysis of the immunogenic potential of the proteins. 2014. 112 f. Tese (Doutorado em Análises quantitativas e moleculares do Genoma; Biologia das células e dos tecidos) - Universidade Federal de Viçosa, Viçosa, 2014.http://locus.ufv.br/handle/123456789/276Dengue 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.A dengue é a principal arbovirose que acomete humanos da atualidade. Com o número de casos cada vez maior é essencial a produção em larga escala de antígenos para o desenvolvimento de kits de diagnóstico rápidos para a detecção de pacientes infectados pelos vírus e a consequente intervenção médica adequada para os mesmos. Além disso, não há vacina eficaz disponível contra esta moléstia que acomete grande parte da população mundial. Assim, neste trabalho nós expressamos as proteínas prM e E do vírus dengue-3 em leveduras Pichia pastoris KM71H. As proteínas foram produzidas de forma solúvel no sobrenadante da cultura sob a forma de vírus- like particles (VLPs). O sobrenadante da cultura de leveduras foi submetido à purificação de proteínas através de cromatografia de afinidade usando uma coluna de níquel, porém sem êxito. Assim, o mesmo foi submetido a precipitações com concentrações de saturação crescentes de sulfato de amônio. Após confirmação por Western Blot, a fração de 20% de sulfato de amônio foi utilizada como antígeno em teste de ELISA indireto utilizando soros de pacientes, fornecendo sensibilidade de 73,91% e especificidade de 82,61%. Já a fração proveniente da precipitação com 80% mostrou-se mais satisfatória, fornecendo um teste com 82,61% de sensibilidade e 89,25% de especificidade para a detecção de anticorpos IgM específicos. Ainda, a fração e 80% foi utilizada para a pesquisa de anticorpos IgG específicos contra o vírus, fornecendo 76,09% de sensibilidade e 84,78% de especificidade. As frações de 20% e 80% foram avaliadas também quanto à sua capacidade de gerar respostas imunes contra o vírus dengue-3 em camundongos. Foi observada uma indução de resposta imune Th2, verificado pelo aumento da produção de IL-4 e IL-10 quando as células dos baços dos animais previamente imunizados foram estimuladas com o vírus dengue-3. Também foi induzida a produção anticorpos específicos nestes animais, mostrado através de imunofluorescência indireta utilizando-se os soros dos camundongos imunizados. Além disso, foi determinada a capacidade neutralizante de tais anticorpos, com um título de 512 de anticorpos neutralizantes. Também foi detectado nos animais imunizados com as proteínas recombinantes um aumento na população de células TCD4 ativadas e TCD4 de memória central. Dessa forma, a metodologia aqui proposta mostrou-se promissora para a obtenção de antígenos dos vírus dengue na busca por um candidato vacinal eficaz e criação de testes diagnósticos rápidos e baratos, o que é de grande valia uma vez que grande parte das áreas atingidas pela dengue são economicamente negligenciadas.Coordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de ViçosaDoutorado em Biologia Celular e EstruturalUFVBRAnálises quantitativas e moleculares do Genoma; Biologia das células e dos tecidosDengue - VacinaDiagnósticoPichia pastorisDengue - VascinadiagnosisPichia pastorisCNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::NUTRICAO E ALIMENTACAO ANIMAL::AVALIACAO DE ALIMENTOS PARA ANIMAISExpressão 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 proteínasExpression of synthetic genes prM and E of dengue-3 virus in Pichia pastoris: evaluation of use in dengue diagnosis and analysis of the immunogenic potential of the proteinsinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf1998515https://locus.ufv.br//bitstream/123456789/276/1/texto%20completo.pdfa8ed258df0f941a684c6cc18dad364daMD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain165820https://locus.ufv.br//bitstream/123456789/276/2/texto%20completo.pdf.txt8c6cff782dae72e77821cf601c21f43aMD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3676https://locus.ufv.br//bitstream/123456789/276/3/texto%20completo.pdf.jpg5cd04a0de265844449c640a08732cc68MD53123456789/2762016-04-06 23:01:19.443oai:locus.ufv.br:123456789/276Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-07T02:01:19LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv 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
dc.title.alternative.eng.fl_str_mv Expression of synthetic genes prM and E of dengue-3 virus in Pichia pastoris: evaluation of use in dengue diagnosis and analysis of the immunogenic potential of the proteins
title 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
spellingShingle 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
Teixeira, Michelle Dias de Oliveira
Dengue - Vacina
Diagnóstico
Pichia pastoris
Dengue - Vascina
diagnosis
Pichia pastoris
CNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::NUTRICAO E ALIMENTACAO ANIMAL::AVALIACAO DE ALIMENTOS PARA ANIMAIS
title_short 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_sort 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
author Teixeira, Michelle Dias de Oliveira
author_facet Teixeira, Michelle Dias de Oliveira
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://lattes.cnpq.br/0612903958610514
dc.contributor.author.fl_str_mv Teixeira, Michelle Dias de Oliveira
dc.contributor.advisor-co1.fl_str_mv Cardoso, Silvia Almeida
dc.contributor.advisor-co1Lattes.fl_str_mv http://lattes.cnpq.br/6041368188542057
dc.contributor.advisor1.fl_str_mv Paula, Sérgio Oliveira de
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4767540P4
dc.contributor.referee1.fl_str_mv Damaso, Clarissa
dc.contributor.referee1Lattes.fl_str_mv http://lattes.cnpq.br/3900177177421722
dc.contributor.referee2.fl_str_mv Eller, Monique Renon
dc.contributor.referee2Lattes.fl_str_mv http://lattes.cnpq.br/4821785523906789
dc.contributor.referee3.fl_str_mv Siqueira-batista, Rodrigo
dc.contributor.referee3Lattes.fl_str_mv http://lattes.cnpq.br/7992589011048146
dc.contributor.referee4.fl_str_mv Gomes, Andréia Patrícia
dc.contributor.referee4Lattes.fl_str_mv http://lattes.cnpq.br/2722908704781524
contributor_str_mv Cardoso, Silvia Almeida
Paula, Sérgio Oliveira de
Damaso, Clarissa
Eller, Monique Renon
Siqueira-batista, Rodrigo
Gomes, Andréia Patrícia
dc.subject.por.fl_str_mv Dengue - Vacina
Diagnóstico
Pichia pastoris
topic Dengue - Vacina
Diagnóstico
Pichia pastoris
Dengue - Vascina
diagnosis
Pichia pastoris
CNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::NUTRICAO E ALIMENTACAO ANIMAL::AVALIACAO DE ALIMENTOS PARA ANIMAIS
dc.subject.eng.fl_str_mv Dengue - Vascina
diagnosis
Pichia pastoris
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::NUTRICAO E ALIMENTACAO ANIMAL::AVALIACAO DE ALIMENTOS PARA ANIMAIS
description 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.
publishDate 2014
dc.date.issued.fl_str_mv 2014-09-09
dc.date.accessioned.fl_str_mv 2015-03-26T12:10:47Z
dc.date.available.fl_str_mv 2015-01-16
2015-03-26T12:10:47Z
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dc.identifier.citation.fl_str_mv TEIXEIRA, Michelle Dias de Oliveira. Expression of synthetic genes prM and E of dengue-3 virus in Pichia pastoris: evaluation of use in dengue diagnosis and analysis of the immunogenic potential of the proteins. 2014. 112 f. Tese (Doutorado em Análises quantitativas e moleculares do Genoma; Biologia das células e dos tecidos) - Universidade Federal de Viçosa, Viçosa, 2014.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/276
identifier_str_mv TEIXEIRA, Michelle Dias de Oliveira. Expression of synthetic genes prM and E of dengue-3 virus in Pichia pastoris: evaluation of use in dengue diagnosis and analysis of the immunogenic potential of the proteins. 2014. 112 f. Tese (Doutorado em Análises quantitativas e moleculares do Genoma; Biologia das células e dos tecidos) - Universidade Federal de Viçosa, Viçosa, 2014.
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dc.publisher.country.fl_str_mv BR
dc.publisher.department.fl_str_mv Análises quantitativas e moleculares do Genoma; Biologia das células e dos tecidos
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