Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais

Detalhes bibliográficos
Autor(a) principal: Santos, Paula de Souza
Data de Publicação: 2011
Tipo de documento: Tese
Idioma: por
Título da fonte: Repositório Institucional da UFU
Texto Completo: https://repositorio.ufu.br/handle/123456789/15730
https://doi.org/10.14393/ufu.te.2011.36
Resumo: Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequences SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized; one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system.
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spelling Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinaisAnaplasma marginalePhage DisplayEpitopoBioeletrodoPeptídeosAnaplasmose - VacinaAnaplasmose - DiagnósticoEpitopeBioelectrodeCNPQ::CIENCIAS BIOLOGICAS::GENETICAAnaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequences SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized; one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system.Doutor em Genética e BioquímicaAnaplasmose, uma infecção intraeritrocitária obrigatória de bovinos, causada pela Anaplasma marginale, causa severa anemia e alta taxa de aborto, resultando em perda significativa para a indústria de laticínios e carne. O diagnóstico clínico é baseado nos sintomas e exames laboratorias confirmatórios são necessários. Atualmente, todos os ensaios de diagnósticos têm sido desenvolvidos com ELISA indireto de antígenos totais baseado em epítopos múltiplos. Em uma investigação pioneira demonstramos o uso de motivos críticos de um epítopo como biomarcador para a aplicação em imunossensores. Mimotopos do epítopo funcional da proteína MSP1a foram obtidos por meio de Phage Display, após três ciclos de seleção, de uma biblioteca randômica de peptídeos 12-mer contra o anticorpo monoclonal 15D2. Trinta e nove clones foram selecionados aleatoriamente, sequenciados, traduzidos e alinhados com a sequência nativa. Foi obtida a sequência consenso SxSSQSEASTSSQLGA, que está localizada na extremidade C-terminal do motivo repetitivo de 28-aa da proteína MSP1a, mas o alinhamento e a variação das sequências entre os mimotopos nos permitiu mapear o motivo crítico STSSxL dentro da sequência consenso. Com base nesses resultados, dois peptídeos foram quimicamente sintetizados; um baseado no motivo crítico (STSSQL, Am1) e o outro baseado na sequência consenso alinhada com o epítopo nativo (SEASTSSQLGA, Am2). Soro de 24 animais infectados e 52 animais saudáveis foram testados por ELISA quanto à reatividade contra Am1 e Am2, que apresentou sensibilidades de 96% e 100%, respectivamente. O peptide Am1 foi incorporado a um bioeletrodo (grafite modificado com ácido poli-3-hidroxyfenilacético) e a detecção direta de soro foi demonstrada por impedância, voltametria de pulso diferencial e microscopia de força atômica. O sistema de sensor eletroquímico provou ser altamente eficaz em soro de animais positivos de negativos. Este imunossensor foi altamente sensível e seletivo para IgG positiva, contaminantes não afetaram as medições, e foram baseados e um sistema simples, rápido e reprodutível.Universidade Federal de UberlândiaBRPrograma de Pós-graduação em Genética e BioquímicaCiências BiológicasUFUGoulart Filho, Luiz Ricardohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781012P8Szabó, Matias Pablo Juanhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4786211E0Azevedo, Vasco Ariston de Carvalhohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787259E8Vidotto, OdilonMineo, Tiago Wilson Patriarcahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4762379Y6Santos, Paula de Souza2016-06-22T18:43:25Z2012-04-022016-06-22T18:43:25Z2011-10-28info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfapplication/pdfSANTOS, Paula de Souza. Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais. 2011. 96 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2011. DOI https://doi.org/10.14393/ufu.te.2011.36https://repositorio.ufu.br/handle/123456789/15730https://doi.org/10.14393/ufu.te.2011.36porinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFUinstname:Universidade Federal de Uberlândia (UFU)instacron:UFU2022-12-15T19:05:05Zoai:repositorio.ufu.br:123456789/15730Repositório InstitucionalONGhttp://repositorio.ufu.br/oai/requestdiinf@dirbi.ufu.bropendoar:2022-12-15T19:05:05Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU)false
dc.title.none.fl_str_mv Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais
title Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais
spellingShingle Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais
Santos, Paula de Souza
Anaplasma marginale
Phage Display
Epitopo
Bioeletrodo
Peptídeos
Anaplasmose - Vacina
Anaplasmose - Diagnóstico
Epitope
Bioelectrode
CNPQ::CIENCIAS BIOLOGICAS::GENETICA
title_short Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais
title_full Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais
title_fullStr Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais
title_full_unstemmed Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais
title_sort Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais
author Santos, Paula de Souza
author_facet Santos, Paula de Souza
author_role author
dc.contributor.none.fl_str_mv Goulart Filho, Luiz Ricardo
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781012P8
Szabó, Matias Pablo Juan
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4786211E0
Azevedo, Vasco Ariston de Carvalho
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787259E8
Vidotto, Odilon
Mineo, Tiago Wilson Patriarca
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4762379Y6
dc.contributor.author.fl_str_mv Santos, Paula de Souza
dc.subject.por.fl_str_mv Anaplasma marginale
Phage Display
Epitopo
Bioeletrodo
Peptídeos
Anaplasmose - Vacina
Anaplasmose - Diagnóstico
Epitope
Bioelectrode
CNPQ::CIENCIAS BIOLOGICAS::GENETICA
topic Anaplasma marginale
Phage Display
Epitopo
Bioeletrodo
Peptídeos
Anaplasmose - Vacina
Anaplasmose - Diagnóstico
Epitope
Bioelectrode
CNPQ::CIENCIAS BIOLOGICAS::GENETICA
description Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequences SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized; one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system.
publishDate 2011
dc.date.none.fl_str_mv 2011-10-28
2012-04-02
2016-06-22T18:43:25Z
2016-06-22T18:43:25Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
format doctoralThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv SANTOS, Paula de Souza. Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais. 2011. 96 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2011. DOI https://doi.org/10.14393/ufu.te.2011.36
https://repositorio.ufu.br/handle/123456789/15730
https://doi.org/10.14393/ufu.te.2011.36
identifier_str_mv SANTOS, Paula de Souza. Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais. 2011. 96 f. Tese (Doutorado em Ciências Biológicas) - Universidade Federal de Uberlândia, Uberlândia, 2011. DOI https://doi.org/10.14393/ufu.te.2011.36
url https://repositorio.ufu.br/handle/123456789/15730
https://doi.org/10.14393/ufu.te.2011.36
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 application/pdf
application/pdf
dc.publisher.none.fl_str_mv Universidade Federal de Uberlândia
BR
Programa de Pós-graduação em Genética e Bioquímica
Ciências Biológicas
UFU
publisher.none.fl_str_mv Universidade Federal de Uberlândia
BR
Programa de Pós-graduação em Genética e Bioquímica
Ciências Biológicas
UFU
dc.source.none.fl_str_mv reponame:Repositório Institucional da UFU
instname:Universidade Federal de Uberlândia (UFU)
instacron:UFU
instname_str Universidade Federal de Uberlândia (UFU)
instacron_str UFU
institution UFU
reponame_str Repositório Institucional da UFU
collection Repositório Institucional da UFU
repository.name.fl_str_mv Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU)
repository.mail.fl_str_mv diinf@dirbi.ufu.br
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