Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais
Autor(a) principal: | |
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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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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 |
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info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
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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 |
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Universidade Federal de Uberlândia (UFU) |
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UFU |
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UFU |
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Repositório Institucional da UFU |
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Repositório Institucional da UFU |
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Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU) |
repository.mail.fl_str_mv |
diinf@dirbi.ufu.br |
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