Clonagem e Expressão da Proteína gp19 de Ehrlichia canis

Detalhes bibliográficos
Autor(a) principal: Brum, Fernanda Antunes
Data de Publicação: 2010
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Repositório Institucional da UFPel - Guaiaca
Texto Completo: http://repositorio.ufpel.edu.br/handle/ri/2346
Resumo: The Ehrlichia canis is a canine monocytic ehrlichiosis responsible for (EMC). The incubation period of EMC is 8 to 20 days, the disease has three phases: acute, subclinical and chronic. The species E. canis is transmitted to the dog and the man by the tick Rhipicephalus sanguineus. It is diagnosed by blood smear, serological or polymerase chain reaction (PCR), with the immunofluorescence method used. Recently E. canis was described as being capable of causing severe disease in humans, with death cases, mainly children and elderly. The gp 19 protein is an important immunodominant antigen, it induces rapid immune response in dogs. The similarity between the geographically distinct samples suggests that the gp19 protein can be used for testing the diagnostic immunoassays, as well as vaccination programs, because this protein is specific for E. canis and thus not have cross reactions with other genera of Ehrlichia.Este study aimed to clone and express the glycoprotein 19 of Ehrlichia canis in Escherichia coli for use as immunobiological, rapid and accurate detection of this disease. The gp 19 gene was amplified by polymerase chain reaction (PCR) using specific primers containing sites for restriction enzymes. The PCR product after digestion, was purified and cloned into a vector and inserted into E. pAE coli TOP 10 competent by electroporation. Of the identified clones was extracted plasmid, which was digested with restriction enzymes to confirm the presence of the insert. After selection of recombinant clones containing the gene linked to the vector, this was purified by heat shock and inserted in expression strain E. coli Star, cultured and induced to express the protein gp19.
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spelling 2014-08-20T14:31:33Z2011-03-252014-08-20T14:31:33Z2010-12-23BRUM, Fernanda Antunes. Cloning and Expression of the gp19 protein of Ehrlichia canis. 2010. 10 f. Dissertação (Mestrado em Biologia) - Universidade Federal de Pelotas, Pelotas, 2010.http://repositorio.ufpel.edu.br/handle/ri/2346The Ehrlichia canis is a canine monocytic ehrlichiosis responsible for (EMC). The incubation period of EMC is 8 to 20 days, the disease has three phases: acute, subclinical and chronic. The species E. canis is transmitted to the dog and the man by the tick Rhipicephalus sanguineus. It is diagnosed by blood smear, serological or polymerase chain reaction (PCR), with the immunofluorescence method used. Recently E. canis was described as being capable of causing severe disease in humans, with death cases, mainly children and elderly. The gp 19 protein is an important immunodominant antigen, it induces rapid immune response in dogs. The similarity between the geographically distinct samples suggests that the gp19 protein can be used for testing the diagnostic immunoassays, as well as vaccination programs, because this protein is specific for E. canis and thus not have cross reactions with other genera of Ehrlichia.Este study aimed to clone and express the glycoprotein 19 of Ehrlichia canis in Escherichia coli for use as immunobiological, rapid and accurate detection of this disease. The gp 19 gene was amplified by polymerase chain reaction (PCR) using specific primers containing sites for restriction enzymes. The PCR product after digestion, was purified and cloned into a vector and inserted into E. pAE coli TOP 10 competent by electroporation. Of the identified clones was extracted plasmid, which was digested with restriction enzymes to confirm the presence of the insert. After selection of recombinant clones containing the gene linked to the vector, this was purified by heat shock and inserted in expression strain E. coli Star, cultured and induced to express the protein gp19.A Ehrlichia canis é a responsável pela erliquiose monocitica canina (EMC). O período de incubação da EMC é de 8 a 20 dias; a doença apresenta três fases: aguda, subclínica e crônica. As espécies de E. canis são transmitidas para o cão e para o homem pelo carrapato da espécie Rhipicephalus sanguineus. O diagnóstico é realizado através de esfregaços sanguíneos, métodos sorológicos ou reação em cadeia da polimerase (PCR), sendo a imunofluorescencia o método mais utilizado. Recentemente E. canis, foi descrita como sendo capaz de causar doença grave em humanos, com casos de óbito principalmente em crianças e idosos. A proteína gp 19 é um importante antígeno imunodominante, pois induz rápida resposta imunológica nos cães. A similaridade entre as amostras geograficamente distintas sugere que a proteína gp19 possa ser usada para ensaios de imunoenzimáticos de diagnóstico, bem como em programas vacinais, pois esta proteína é especifica para E. canis não tendo assim reações cruzadas com outros gêneros de Ehrlichia. O gene gp 19 foi amplificado pela reação da polimerase em cadeia (PCR) utilizando oligonucleotídeos iniciadores específicos contendo sítios para enzimas de restrição. O produto da PCR, após digestão, foi purificado e clonado no vetor pAE e inserido em E. coli TOP 10 competente por eletroporação. Dos clones identificados extraiu-se o plasmídio, o qual foi digerido com as enzimas de restrição para comprovar a presença do inserto. Após seleção dos clones recombinantes contendo o gene ligado ao vetor, este foi purificado e inserido por choque térmico na cepa de expressão E. coli Star cultivado e induzido para expressar a proteína gp19. Esta expressão foi verificada por gel de SDS-Page 12% e confirmada por Dot-Blot.application/pdfporUniversidade Federal de PelotasPrograma de Pós-Graduação em ParasitologiaUFPelBRBiologiaEhrlichia canisExpressão de proteínaRhipicephalus sanguineurProtein expressionRhipicephalus sanguineusCNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIAClonagem e Expressão da Proteína gp19 de Ehrlichia canisCloning and Expression of the gp19 protein of Ehrlichia canisinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesishttp://lattes.cnpq.br/1240795225692026http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763076J5Leite, Fabio Pereira LeivasBrum, Fernanda Antunesinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFPel - Guaiacainstname:Universidade Federal de Pelotas (UFPEL)instacron:UFPELORIGINALdissertacao_fernanda_antunes_brum.pdfapplication/pdf139752http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/2346/1/dissertacao_fernanda_antunes_brum.pdf989f0c94f1c779f13cc72237d9ab8552MD51open accessTEXTdissertacao_fernanda_antunes_brum.pdf.txtdissertacao_fernanda_antunes_brum.pdf.txtExtracted Texttext/plain11217http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/2346/2/dissertacao_fernanda_antunes_brum.pdf.txt7ec351972bbfee7a1a95d830fb46324bMD52open accessTHUMBNAILdissertacao_fernanda_antunes_brum.pdf.jpgdissertacao_fernanda_antunes_brum.pdf.jpgGenerated Thumbnailimage/jpeg1795http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/2346/3/dissertacao_fernanda_antunes_brum.pdf.jpge39a98e5e3b7d4d36749bad63b706513MD53open access123456789/23462019-09-27 10:45:12.974open accessoai:guaiaca.ufpel.edu.br:123456789/2346Repositório InstitucionalPUBhttp://repositorio.ufpel.edu.br/oai/requestrippel@ufpel.edu.br || repositorio@ufpel.edu.br || aline.batista@ufpel.edu.bropendoar:2019-09-27T13:45:12Repositório Institucional da UFPel - Guaiaca - Universidade Federal de Pelotas (UFPEL)false
dc.title.por.fl_str_mv Clonagem e Expressão da Proteína gp19 de Ehrlichia canis
dc.title.alternative.eng.fl_str_mv Cloning and Expression of the gp19 protein of Ehrlichia canis
title Clonagem e Expressão da Proteína gp19 de Ehrlichia canis
spellingShingle Clonagem e Expressão da Proteína gp19 de Ehrlichia canis
Brum, Fernanda Antunes
Ehrlichia canis
Expressão de proteína
Rhipicephalus sanguineur
Protein expression
Rhipicephalus sanguineus
CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA
title_short Clonagem e Expressão da Proteína gp19 de Ehrlichia canis
title_full Clonagem e Expressão da Proteína gp19 de Ehrlichia canis
title_fullStr Clonagem e Expressão da Proteína gp19 de Ehrlichia canis
title_full_unstemmed Clonagem e Expressão da Proteína gp19 de Ehrlichia canis
title_sort Clonagem e Expressão da Proteína gp19 de Ehrlichia canis
author Brum, Fernanda Antunes
author_facet Brum, Fernanda Antunes
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://lattes.cnpq.br/1240795225692026
dc.contributor.advisorLattes.por.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763076J5
dc.contributor.advisor1.fl_str_mv Leite, Fabio Pereira Leivas
dc.contributor.author.fl_str_mv Brum, Fernanda Antunes
contributor_str_mv Leite, Fabio Pereira Leivas
dc.subject.por.fl_str_mv Ehrlichia canis
Expressão de proteína
Rhipicephalus sanguineur
topic Ehrlichia canis
Expressão de proteína
Rhipicephalus sanguineur
Protein expression
Rhipicephalus sanguineus
CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA
dc.subject.eng.fl_str_mv Protein expression
Rhipicephalus sanguineus
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS BIOLOGICAS::PARASITOLOGIA
description The Ehrlichia canis is a canine monocytic ehrlichiosis responsible for (EMC). The incubation period of EMC is 8 to 20 days, the disease has three phases: acute, subclinical and chronic. The species E. canis is transmitted to the dog and the man by the tick Rhipicephalus sanguineus. It is diagnosed by blood smear, serological or polymerase chain reaction (PCR), with the immunofluorescence method used. Recently E. canis was described as being capable of causing severe disease in humans, with death cases, mainly children and elderly. The gp 19 protein is an important immunodominant antigen, it induces rapid immune response in dogs. The similarity between the geographically distinct samples suggests that the gp19 protein can be used for testing the diagnostic immunoassays, as well as vaccination programs, because this protein is specific for E. canis and thus not have cross reactions with other genera of Ehrlichia.Este study aimed to clone and express the glycoprotein 19 of Ehrlichia canis in Escherichia coli for use as immunobiological, rapid and accurate detection of this disease. The gp 19 gene was amplified by polymerase chain reaction (PCR) using specific primers containing sites for restriction enzymes. The PCR product after digestion, was purified and cloned into a vector and inserted into E. pAE coli TOP 10 competent by electroporation. Of the identified clones was extracted plasmid, which was digested with restriction enzymes to confirm the presence of the insert. After selection of recombinant clones containing the gene linked to the vector, this was purified by heat shock and inserted in expression strain E. coli Star, cultured and induced to express the protein gp19.
publishDate 2010
dc.date.issued.fl_str_mv 2010-12-23
dc.date.available.fl_str_mv 2011-03-25
2014-08-20T14:31:33Z
dc.date.accessioned.fl_str_mv 2014-08-20T14:31:33Z
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dc.identifier.citation.fl_str_mv BRUM, Fernanda Antunes. Cloning and Expression of the gp19 protein of Ehrlichia canis. 2010. 10 f. Dissertação (Mestrado em Biologia) - Universidade Federal de Pelotas, Pelotas, 2010.
dc.identifier.uri.fl_str_mv http://repositorio.ufpel.edu.br/handle/ri/2346
identifier_str_mv BRUM, Fernanda Antunes. Cloning and Expression of the gp19 protein of Ehrlichia canis. 2010. 10 f. Dissertação (Mestrado em Biologia) - Universidade Federal de Pelotas, Pelotas, 2010.
url http://repositorio.ufpel.edu.br/handle/ri/2346
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