Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti

Detalhes bibliográficos
Ano de defesa: 2009
Autor(a) principal: Soares, Tatiane Sanches [UNIFESP]
Orientador(a): Não Informado pela instituição
Banca de defesa: Não Informado pela instituição
Tipo de documento: Dissertação
Tipo de acesso: Acesso aberto
dARK ID: ark:/48912/001300002gkhg
Idioma: por
Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
Programa de Pós-Graduação: Não Informado pela instituição
Departamento: Não Informado pela instituição
País: Não Informado pela instituição
Palavras-chave em Português:
Enzimas digestivas
Inibidores de serinoproteases
Mosquitos
Aedes aegypti
Serina proteases
Culicidae
Enzimas
Larva
Tripsina
Trypsin-like enzymes
Enzymes
Aedes
Link de acesso: http://repositorio.unifesp.br/handle/11600/9600
Resumo: Aedes aegypti is the most important vector of human arboviral diseases and it is responsible for dengue and urban yellow fever transmissions. Trypsin-like enzymes plays an important role in the Ae. aegypti adult and larval life stages digestion. In the present work, we identified the two major trypsin-like enzymes of Ae. aegypti larval midgut through the trypsin cDNA fragments library construction. They are AAEL005607 and AAEL006371, with expression frequencies of 29.3% and 20%, respectively. Semi quantitative PCR analysis showed that the AAEL005607 was transcripted in all larval instars, but AAEL006371 appeared only in 3rd and 4th larval instars. In order to confirm the transcription data, trypsin-like enzymes from 4th instar larvae of Ae. aegypti midgut were purified by affinity, ionic exchange and reversedphase chromatographies. Purified trypsin presented molecular mass of 28 kDa by SDS-PAGE. Its partial amino acid sequence allowed us to suggest that the trypsin activity is encoding by AAEL005607 sequence. The purified trypsin (AAEL005607) showed Km value of 36.4 μM for Tosyl-Gly-Pro-Arg-pNa substrate and was strongly inhibited by AaTI and HiTI, both trypsin inhibitors, with Ki of 0.94 pM and 160 pM, respectively. In conclusion, for the first time, the major digestive enzyme of 4th larval instar of Ae. aegypti was identified and characterized.
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spelling Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegyptiMolecular studies of trypsin-like enzymes present in midgut of Aedes aegypti larvaeEnzimas digestivasInibidores de serinoproteasesMosquitosAedes aegyptiSerina proteasesCulicidaeEnzimasLarvaTripsinaLarvaTrypsin-like enzymesEnzymesAedesAedes aegypti is the most important vector of human arboviral diseases and it is responsible for dengue and urban yellow fever transmissions. Trypsin-like enzymes plays an important role in the Ae. aegypti adult and larval life stages digestion. In the present work, we identified the two major trypsin-like enzymes of Ae. aegypti larval midgut through the trypsin cDNA fragments library construction. They are AAEL005607 and AAEL006371, with expression frequencies of 29.3% and 20%, respectively. Semi quantitative PCR analysis showed that the AAEL005607 was transcripted in all larval instars, but AAEL006371 appeared only in 3rd and 4th larval instars. In order to confirm the transcription data, trypsin-like enzymes from 4th instar larvae of Ae. aegypti midgut were purified by affinity, ionic exchange and reversedphase chromatographies. Purified trypsin presented molecular mass of 28 kDa by SDS-PAGE. Its partial amino acid sequence allowed us to suggest that the trypsin activity is encoding by AAEL005607 sequence. The purified trypsin (AAEL005607) showed Km value of 36.4 μM for Tosyl-Gly-Pro-Arg-pNa substrate and was strongly inhibited by AaTI and HiTI, both trypsin inhibitors, with Ki of 0.94 pM and 160 pM, respectively. In conclusion, for the first time, the major digestive enzyme of 4th larval instar of Ae. aegypti was identified and characterized.O Aedes aegypti é o vetor mais importante de arboviroses humana sendo responsável pelas transmissões de dengue e febre amarela urbana. As enzimas tipo tripsina apresentam um importante papel na digestão de estádios de vida larval e adulto de Ae. aegypti. No presente trabalho, nós identificamos as duas enzimas tipo tripsina majoritárias do intestino médio larval através da construção de uma biblioteca de fragmentos de cDNA de tripsina. Elas são AAEL005607 e AAEL006371, com frequências de expressão de 29,3% e 20%, respectivamente. Análises por PCR semi-quantitativo mostraram que a tripsina AAEL005607 foi transcrita em todos os instars larval, mas a tripsina AAEL006371 apareceu somente nos 3º e 4º instar larvais. A fim de confirmar os dados de transcrição, enzimas tipo tripsina do intestino médio de larvas de 4º instar foram purificadas por cromatografias de afinidade, troca iônica e fase reversa. A tripsina purificada apresentou massa molecular de 28 kDa por SDS-PAGE. Sua sequência de aminoácidos parcial nos permitiu sugerir que a atividade de tripsina é codificada pela sequência AAEL005607. A tripsina purificada (AAEL005607) exibiu um valor de Km de 36,4 μM para o substrato Tosyl-Gly-Pro-Arg-pNa e foi fortemente inibida por AaTI e HiTI, ambos inibidores de tripsina, com valores de Ki de 0,94 pM e 160 pM, respectivamente. Em conclusão, pela primeira vez, a enzima digestiva majoritária de 4º instar larval de Ae. aegypti foi purificada e caracterizada.TEDEBV UNIFESP: Teses e dissertaçõesUniversidade Federal de São Paulo (UNIFESP)Tanaka, Aparecida Sadae [UNIFESP]Universidade Federal de São Paulo (UNIFESP)Soares, Tatiane Sanches [UNIFESP]2015-07-22T20:50:11Z2015-07-22T20:50:11Z2009-07-29info:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/publishedVersion106 p.application/pdfSOARES, Tatiane Sanches. Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti. 2009. 106 f. Dissertação (Mestrado) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2009.Publico-128.pdfhttp://repositorio.unifesp.br/handle/11600/9600ark:/48912/001300002gkhgporinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP2024-08-05T18:46:41Zoai:repositorio.unifesp.br:11600/9600Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestbiblioteca.csp@unifesp.bropendoar:34652024-08-05T18:46:41Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false
dc.title.none.fl_str_mv Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti
Molecular studies of trypsin-like enzymes present in midgut of Aedes aegypti larvae
title Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti
spellingShingle Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti
Soares, Tatiane Sanches [UNIFESP]
Enzimas digestivas
Inibidores de serinoproteases
Mosquitos
Aedes aegypti
Serina proteases
Culicidae
Enzimas
Larva
Tripsina
Larva
Trypsin-like enzymes
Enzymes
Aedes
title_short Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti
title_full Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti
title_fullStr Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti
title_full_unstemmed Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti
title_sort Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti
author Soares, Tatiane Sanches [UNIFESP]
author_facet Soares, Tatiane Sanches [UNIFESP]
author_role author
dc.contributor.none.fl_str_mv Tanaka, Aparecida Sadae [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
dc.contributor.author.fl_str_mv Soares, Tatiane Sanches [UNIFESP]
dc.subject.por.fl_str_mv Enzimas digestivas
Inibidores de serinoproteases
Mosquitos
Aedes aegypti
Serina proteases
Culicidae
Enzimas
Larva
Tripsina
Larva
Trypsin-like enzymes
Enzymes
Aedes
topic Enzimas digestivas
Inibidores de serinoproteases
Mosquitos
Aedes aegypti
Serina proteases
Culicidae
Enzimas
Larva
Tripsina
Larva
Trypsin-like enzymes
Enzymes
Aedes
description Aedes aegypti is the most important vector of human arboviral diseases and it is responsible for dengue and urban yellow fever transmissions. Trypsin-like enzymes plays an important role in the Ae. aegypti adult and larval life stages digestion. In the present work, we identified the two major trypsin-like enzymes of Ae. aegypti larval midgut through the trypsin cDNA fragments library construction. They are AAEL005607 and AAEL006371, with expression frequencies of 29.3% and 20%, respectively. Semi quantitative PCR analysis showed that the AAEL005607 was transcripted in all larval instars, but AAEL006371 appeared only in 3rd and 4th larval instars. In order to confirm the transcription data, trypsin-like enzymes from 4th instar larvae of Ae. aegypti midgut were purified by affinity, ionic exchange and reversedphase chromatographies. Purified trypsin presented molecular mass of 28 kDa by SDS-PAGE. Its partial amino acid sequence allowed us to suggest that the trypsin activity is encoding by AAEL005607 sequence. The purified trypsin (AAEL005607) showed Km value of 36.4 μM for Tosyl-Gly-Pro-Arg-pNa substrate and was strongly inhibited by AaTI and HiTI, both trypsin inhibitors, with Ki of 0.94 pM and 160 pM, respectively. In conclusion, for the first time, the major digestive enzyme of 4th larval instar of Ae. aegypti was identified and characterized.
publishDate 2009
dc.date.none.fl_str_mv 2009-07-29
2015-07-22T20:50:11Z
2015-07-22T20:50:11Z
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv SOARES, Tatiane Sanches. Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti. 2009. 106 f. Dissertação (Mestrado) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2009.
Publico-128.pdf
http://repositorio.unifesp.br/handle/11600/9600
dc.identifier.dark.fl_str_mv ark:/48912/001300002gkhg
identifier_str_mv SOARES, Tatiane Sanches. Estudos moleculares de enzimas do tipo tripsina presentes no intestino médio de larvas de Aedes aegypti. 2009. 106 f. Dissertação (Mestrado) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2009.
Publico-128.pdf
ark:/48912/001300002gkhg
url http://repositorio.unifesp.br/handle/11600/9600
dc.language.iso.fl_str_mv por
language por
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 106 p.
application/pdf
dc.publisher.none.fl_str_mv Universidade Federal de São Paulo (UNIFESP)
publisher.none.fl_str_mv Universidade Federal de São Paulo (UNIFESP)
dc.source.none.fl_str_mv reponame:Repositório Institucional da UNIFESP
instname:Universidade Federal de São Paulo (UNIFESP)
instacron:UNIFESP
instname_str Universidade Federal de São Paulo (UNIFESP)
instacron_str UNIFESP
institution UNIFESP
reponame_str Repositório Institucional da UNIFESP
collection Repositório Institucional da UNIFESP
repository.name.fl_str_mv Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)
repository.mail.fl_str_mv biblioteca.csp@unifesp.br
_version_ 1848498011886321664

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