Expressão e caracterização de um fragmento da glicoproteína e do herpesvírus bovino tipo 1 e uso em um teste sorológico diferencial
Ano de defesa: | 2012 |
---|---|
Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | , |
Tipo de documento: | Dissertação |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade Federal de Santa Maria
|
Programa de Pós-Graduação: |
Programa de Pós-Graduação em Medicina Veterinária
|
Departamento: |
Medicina Veterinária
|
País: |
BR
|
Palavras-chave em Português: | |
Palavras-chave em Inglês: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://repositorio.ufsm.br/handle/1/10116 |
Resumo: | Bovine herpesvirus type 1 (BoHV-1) is distributed worldwide and produces high economic losses to the in livestock industry. BoHV-1 infection causes respiratory, reproductive and may also be associated with neurological signs. There are several tests that can diagnose the infection, however, serological techniques currently used are not able to differentiate antibodies produced by vaccination from those produced in response to natural infection. What is sought is a mean to differentiate vaccinated animals of those infected by the field strain. Vaccines with deletion in the glycoprotein E (gE) gene have been developed for this purpose. However, this also requires the development of tests capable to differentiate the serological response between infected and vaccinated animals. To this end, a 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of the BoHV-1 gE gene - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector pET16b. A soluble protein of approximately 25 kDa was purified from lysates of transformed E. coli. The recombinant protein was detected in Western blot (WB) by anti-6-his tag and anti BoHV-1 gE monoclonal antibodies. Antibodies present in the sera of cattle infected with BoHV-1 and BoHV-5 reacted specifically with the 25 kDa recombinant protein in WB. Moreover, mice immunized with the purified protein developed antibodies that recognized the viral gE in lysates of cell monolayers infected with BoHV-1 and BoHV-5. An indirect ELISA for gE antibodies, based on the expressed protein, was able to differentiate serologically calves vaccinated with a gE-deleted BoHV- 5 strain from calves experimentally infected with BoHV-1 or BoHV-5. These data demonstrate that the antigen retained its immunological properties and, thus, can be used in serological tests for bovine herpesvirus infections. It has a potential use in a indirect ELISA to differentiate naturally infected animals from those vaccinated whit the recombinant, gE-negative strains. |
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2012-09-282012-09-282012-03-05OLIVEIRA, Stephan Alberto Machado de. Expression and characterization of a truncated form of bovine herpesvirus type 1 envelope glycoprotein e and its use in a differential serological test. 2012. 48 f. Dissertação (Mestrado em Medicina Veterinária) - Universidade Federal de Santa Maria, Santa Maria, 2012.http://repositorio.ufsm.br/handle/1/10116Bovine herpesvirus type 1 (BoHV-1) is distributed worldwide and produces high economic losses to the in livestock industry. BoHV-1 infection causes respiratory, reproductive and may also be associated with neurological signs. There are several tests that can diagnose the infection, however, serological techniques currently used are not able to differentiate antibodies produced by vaccination from those produced in response to natural infection. What is sought is a mean to differentiate vaccinated animals of those infected by the field strain. Vaccines with deletion in the glycoprotein E (gE) gene have been developed for this purpose. However, this also requires the development of tests capable to differentiate the serological response between infected and vaccinated animals. To this end, a 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of the BoHV-1 gE gene - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector pET16b. A soluble protein of approximately 25 kDa was purified from lysates of transformed E. coli. The recombinant protein was detected in Western blot (WB) by anti-6-his tag and anti BoHV-1 gE monoclonal antibodies. Antibodies present in the sera of cattle infected with BoHV-1 and BoHV-5 reacted specifically with the 25 kDa recombinant protein in WB. Moreover, mice immunized with the purified protein developed antibodies that recognized the viral gE in lysates of cell monolayers infected with BoHV-1 and BoHV-5. An indirect ELISA for gE antibodies, based on the expressed protein, was able to differentiate serologically calves vaccinated with a gE-deleted BoHV- 5 strain from calves experimentally infected with BoHV-1 or BoHV-5. These data demonstrate that the antigen retained its immunological properties and, thus, can be used in serological tests for bovine herpesvirus infections. It has a potential use in a indirect ELISA to differentiate naturally infected animals from those vaccinated whit the recombinant, gE-negative strains.O herpesvírus bovino tipo 1 (BoHV-1) é um vírus de distribuição mundial e produz grandes prejuízos econômicos em rebanhos de corte e de leite. A infecção pelo BoHV-1 produz manifestações respiratórias, reprodutivas e também pode cursar com sinais nervosos. Existem diversos testes laboratoriais capazes de diagnosticar a infecção. Contudo, as técnicas sorológicas empregadas atualmente não são capazes de diferenciar anticorpos produzidos pela vacinação daqueles produzidos em resposta à infecção natural. Assim, vacinas diferenciais com deleção da glicoproteína E (gE) têm sido desenvolvidas com essa finalidade. No entanto, necessita-se também de testes capazes de diferenciar a produção de anticorpos vacinais dos induzidos pelo vírus vacinal. Com essa finalidade, essa dissertação relata a expressão e caracterização de um fragmento da glicoproteína E do BoHV-1 e seu uso no desenvolvimento e padronização de um ELISA indireto para detecção de anticorpos anti-gE. Um fragmento de 651 nucleotídeos correspondente ao terço amino-terminal (217 aminoácidos) do gene da gE do BoHV-1, que possui uma alta identidade com o homólogo herpesvírus bovino tipo 5 (BoHV-5), foi clonado com proteína de fusão 6xHis-tag em Escherichia coli utilizando vetor de expressão pET16b. Uma proteína solúvel de aproximadamente 25kDa foi purificada a partir de lisados de E. coli transformadas. A proteína recombinante foi detectada por Western blot (WB) por anticorpos monoclonais anti-histidina e anti-gE do BoHV-1. Anticorpos presentes no soro de animais infectados com BoHV-1 e BoHV-5 reagiram especificamente com a proteína recombinante no WB. Além disso, camundongos imunizados com a proteína purificada desenvolveram anticorpos que reconheceram a gE viral proveniente de lisados de monocamadas celulares infectadas com BoHV-1 e BoHV-5. Um ELISA indireto para detecção de anticorpos anti-gE, baseado na proteína expressa, foi capaz de diferenciar sorologicamente animais vacinados com a cepa gE deletada do BoHV-5 dos animais experimentalmente infectados com BoHV-1 ou BoHV-5. Esses resultados demonstram que o antígeno obtido conservou suas características imunológicas e pode ser utilizado na detecção sorológica das infecções por herpesvírus bovinos. Possui potencial para uso em grande escala como antígeno em testes de ELISA para diferenciar animais naturalmente infectados de animais vacinados com a cepas defectivas na gECoordenação de Aperfeiçoamento de Pessoal de Nível Superiorapplication/pdfporUniversidade Federal de Santa MariaPrograma de Pós-Graduação em Medicina VeterináriaUFSMBRMedicina VeterináriaTeste imunoenzimáticoBoHV-1BoHV-5AntígenoProteína recombinanteEnzyme-linked immunosorbent assayAntigenRecombinant proteinCNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIAExpressão e caracterização de um fragmento da glicoproteína e do herpesvírus bovino tipo 1 e uso em um teste sorológico diferencialExpression and characterization of a truncated form of bovine herpesvirus type 1 envelope glycoprotein e and its use in a differential serological testinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisWeiblen, Rudihttp://lattes.cnpq.br/7946350215388090Brum, Mário Celso Sperottohttp://lattes.cnpq.br/9761857774819478Lima, Marcelo dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.dohttp://lattes.cnpq.br/9806223507710114Oliveira, Stephan Alberto Machado de50050000000740050030030050002776f4b-7154-4d1a-9f55-d68a031b94bc9ae87526-5dc5-44bc-a10c-c006f7ae9872f8b3b09a-5476-4345-8c06-4ebd51dad5312aa5f112-79ac-446f-9733-4eb071ee5cccinfo:eu-repo/semantics/openAccessreponame:Manancial - Repositório Digital da UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSMORIGINALOLIVEIRA, STEPHAN ALBERTO MACHADO DE.pdfapplication/pdf966049http://repositorio.ufsm.br/bitstream/1/10116/1/OLIVEIRA%2c%20STEPHAN%20ALBERTO%20MACHADO%20DE.pdf7d74c7e70d45ed795ef60aabd86d1457MD51TEXTOLIVEIRA, STEPHAN ALBERTO MACHADO DE.pdf.txtOLIVEIRA, STEPHAN ALBERTO MACHADO DE.pdf.txtExtracted texttext/plain74578http://repositorio.ufsm.br/bitstream/1/10116/2/OLIVEIRA%2c%20STEPHAN%20ALBERTO%20MACHADO%20DE.pdf.txt8cd60d90676b888b18fbc8dabee9c33eMD52THUMBNAILOLIVEIRA, STEPHAN ALBERTO MACHADO DE.pdf.jpgOLIVEIRA, STEPHAN ALBERTO MACHADO DE.pdf.jpgIM Thumbnailimage/jpeg5226http://repositorio.ufsm.br/bitstream/1/10116/3/OLIVEIRA%2c%20STEPHAN%20ALBERTO%20MACHADO%20DE.pdf.jpg903b24b0ed5e4ad51bb155edac856238MD531/101162022-01-11 10:33:40.062oai:repositorio.ufsm.br:1/10116Repositório Institucionalhttp://repositorio.ufsm.br/PUBhttp://repositorio.ufsm.br/oai/requestopendoar:39132022-01-11T13:33:40Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM)false |
dc.title.por.fl_str_mv |
Expressão e caracterização de um fragmento da glicoproteína e do herpesvírus bovino tipo 1 e uso em um teste sorológico diferencial |
dc.title.alternative.eng.fl_str_mv |
Expression and characterization of a truncated form of bovine herpesvirus type 1 envelope glycoprotein e and its use in a differential serological test |
title |
Expressão e caracterização de um fragmento da glicoproteína e do herpesvírus bovino tipo 1 e uso em um teste sorológico diferencial |
spellingShingle |
Expressão e caracterização de um fragmento da glicoproteína e do herpesvírus bovino tipo 1 e uso em um teste sorológico diferencial Oliveira, Stephan Alberto Machado de Teste imunoenzimático BoHV-1 BoHV-5 Antígeno Proteína recombinante Enzyme-linked immunosorbent assay Antigen Recombinant protein CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
title_short |
Expressão e caracterização de um fragmento da glicoproteína e do herpesvírus bovino tipo 1 e uso em um teste sorológico diferencial |
title_full |
Expressão e caracterização de um fragmento da glicoproteína e do herpesvírus bovino tipo 1 e uso em um teste sorológico diferencial |
title_fullStr |
Expressão e caracterização de um fragmento da glicoproteína e do herpesvírus bovino tipo 1 e uso em um teste sorológico diferencial |
title_full_unstemmed |
Expressão e caracterização de um fragmento da glicoproteína e do herpesvírus bovino tipo 1 e uso em um teste sorológico diferencial |
title_sort |
Expressão e caracterização de um fragmento da glicoproteína e do herpesvírus bovino tipo 1 e uso em um teste sorológico diferencial |
author |
Oliveira, Stephan Alberto Machado de |
author_facet |
Oliveira, Stephan Alberto Machado de |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Weiblen, Rudi |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/7946350215388090 |
dc.contributor.referee1.fl_str_mv |
Brum, Mário Celso Sperotto |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/9761857774819478 |
dc.contributor.referee2.fl_str_mv |
Lima, Marcelo de |
dc.contributor.referee2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/9806223507710114 |
dc.contributor.author.fl_str_mv |
Oliveira, Stephan Alberto Machado de |
contributor_str_mv |
Weiblen, Rudi Brum, Mário Celso Sperotto Lima, Marcelo de |
dc.subject.por.fl_str_mv |
Teste imunoenzimático BoHV-1 BoHV-5 Antígeno Proteína recombinante |
topic |
Teste imunoenzimático BoHV-1 BoHV-5 Antígeno Proteína recombinante Enzyme-linked immunosorbent assay Antigen Recombinant protein CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
dc.subject.eng.fl_str_mv |
Enzyme-linked immunosorbent assay Antigen Recombinant protein |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
description |
Bovine herpesvirus type 1 (BoHV-1) is distributed worldwide and produces high economic losses to the in livestock industry. BoHV-1 infection causes respiratory, reproductive and may also be associated with neurological signs. There are several tests that can diagnose the infection, however, serological techniques currently used are not able to differentiate antibodies produced by vaccination from those produced in response to natural infection. What is sought is a mean to differentiate vaccinated animals of those infected by the field strain. Vaccines with deletion in the glycoprotein E (gE) gene have been developed for this purpose. However, this also requires the development of tests capable to differentiate the serological response between infected and vaccinated animals. To this end, a 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of the BoHV-1 gE gene - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector pET16b. A soluble protein of approximately 25 kDa was purified from lysates of transformed E. coli. The recombinant protein was detected in Western blot (WB) by anti-6-his tag and anti BoHV-1 gE monoclonal antibodies. Antibodies present in the sera of cattle infected with BoHV-1 and BoHV-5 reacted specifically with the 25 kDa recombinant protein in WB. Moreover, mice immunized with the purified protein developed antibodies that recognized the viral gE in lysates of cell monolayers infected with BoHV-1 and BoHV-5. An indirect ELISA for gE antibodies, based on the expressed protein, was able to differentiate serologically calves vaccinated with a gE-deleted BoHV- 5 strain from calves experimentally infected with BoHV-1 or BoHV-5. These data demonstrate that the antigen retained its immunological properties and, thus, can be used in serological tests for bovine herpesvirus infections. It has a potential use in a indirect ELISA to differentiate naturally infected animals from those vaccinated whit the recombinant, gE-negative strains. |
publishDate |
2012 |
dc.date.accessioned.fl_str_mv |
2012-09-28 |
dc.date.available.fl_str_mv |
2012-09-28 |
dc.date.issued.fl_str_mv |
2012-03-05 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
OLIVEIRA, Stephan Alberto Machado de. Expression and characterization of a truncated form of bovine herpesvirus type 1 envelope glycoprotein e and its use in a differential serological test. 2012. 48 f. Dissertação (Mestrado em Medicina Veterinária) - Universidade Federal de Santa Maria, Santa Maria, 2012. |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufsm.br/handle/1/10116 |
identifier_str_mv |
OLIVEIRA, Stephan Alberto Machado de. Expression and characterization of a truncated form of bovine herpesvirus type 1 envelope glycoprotein e and its use in a differential serological test. 2012. 48 f. Dissertação (Mestrado em Medicina Veterinária) - Universidade Federal de Santa Maria, Santa Maria, 2012. |
url |
http://repositorio.ufsm.br/handle/1/10116 |
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por |
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Universidade Federal de Santa Maria |
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UFSM |
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BR |
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Medicina Veterinária |
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Universidade Federal de Santa Maria |
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