Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display
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| Hlavní autor: | |
|---|---|
| Datum vydání: | 2015 |
| Médium: | Doctoral thesis |
| Jazyk: | por |
| Zdroj: | Repositório Institucional da UFG |
| Download full: | http://repositorio.bc.ufg.br/tede/handle/tede/5503 |
Shrnutí: | Member of the Herpesviridae family, subfamily Alphaherpesvirinae, gender Varicellovirus, the bovine herpesvirus 1 (BoHV-1) has been associated with different clinical conditions (respiratory and genital/reproductive diseases) in cattle. There is no standard procedure to control or prevent infections caused by herpesviruses. In this sense, phage display was used to select new glycoprotein mimotopes antigen of BoHV-1 that has potential for use in vaccines and diagnostics. The phage display technique was performed using a linear random peptide library consisting of 12 amino acid residues fused to the protein III of M13 phage (no peptide) against BoHV-1 specific IgGs, purified by affinity chromatography. After three cycles of selection (biopanning) and amplification, 44 clones were isolated and their amino acid sequences were determined by sequencing generating 16 different sequences. ELISA, demonstrating the efficiency of selection from the specific antibodies, confirmed the reactivity of pooled clones. Another ELISA evaluated the individual specificity of the most frequent clones, the M13 phage was used as a negative control. We selected three peptides (B, C and E) with affinity for anti-BoHV-1 antibodies, and the E peptide (pepE), showed to have potential as antigen for antibody detection in a serological test for BoHV-1. Immunization of rabbits with the peptides induced specific production of serum antibodies, but they were not able do neutralize BoHV-1 cell lysis. The in silico analysis of the dodecapeptide E (1DRALYGPTVIDH12) enabled the identification of a new discontinuous epitope on the envelope glycoprotein B (gB Env) of bovine herpesvirus type 1 (BoHV-1). There is a short motif (338YKRD341) within a region of the env gB BoHV-1 with high similarity to motifs shared by dodecapeptide the N-terminal region (5YxARD1) of gB and HSV-1 (326YARD329), wherein the 328Arg residue is described as a target for neutralizing monoclonal antibodies (mAb) for HSV-1 gB. Besides the characterization of an antibody-binding site of the BoHV-1 Env gB, we have demonstrated that the phage-fused peptide has potential use as a reagent for virus diagnosis by phage-ELISA assay, discriminating BoHV-1 positive serum samples from negative ones. |
| _version_ | 1871426407111852032 |
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| author | Almeida, Greyciele Rodrigues de |
| author_browse | Almeida, Greyciele Rodrigues de |
| author_facet | Almeida, Greyciele Rodrigues de |
| author_role | author |
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| collection | Repositório Institucional da UFG |
| contributor_str_mv | Souza, Guilherme Rocha Lino de Brito, Wilia Marta Elsner Diederichsen de Bataus, Luiz Artur Mendes Souza, Guilherme Rocha Lino de Fiaccadori, Fabiola Souza Kipnis, André Cunha, Paulo Henrique Jorge da Oliveira, Cairo Henrique Sousa de |
| dc.contributor.advisor-co1.fl_str_mv | Brito, Wilia Marta Elsner Diederichsen de |
| dc.contributor.advisor-co2.fl_str_mv | Bataus, Luiz Artur Mendes |
| dc.contributor.advisor-co2Lattes.fl_str_mv | http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785339A7 |
| dc.contributor.advisor1.fl_str_mv | Souza, Guilherme Rocha Lino de |
| dc.contributor.advisor1Lattes.fl_str_mv | http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4700389E3 |
| dc.contributor.author.fl_str_mv | Almeida, Greyciele Rodrigues de |
| dc.contributor.authorLattes.fl_str_mv | http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4247180T2 |
| dc.contributor.referee1.fl_str_mv | Souza, Guilherme Rocha Lino de |
| dc.contributor.referee2.fl_str_mv | Fiaccadori, Fabiola Souza |
| dc.contributor.referee3.fl_str_mv | Kipnis, André |
| dc.contributor.referee4.fl_str_mv | Cunha, Paulo Henrique Jorge da |
| dc.contributor.referee5.fl_str_mv | Oliveira, Cairo Henrique Sousa de |
| dc.date.accessioned.fl_str_mv | 2016-04-20T15:16:36Z |
| dc.date.issued.fl_str_mv | 2015-08-24 |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.citation.fl_str_mv | ALMEIDA, G. R. Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display. 2015. 60 f. Tese (Doutorado em Ciência Animal) - Universidade Federal de Goiás, Goiânia, 2015. |
| dc.identifier.uri.fl_str_mv | http://repositorio.bc.ufg.br/tede/handle/tede/5503 |
| dc.language.iso.fl_str_mv | por |
| dc.publisher.country.fl_str_mv | Brasil |
| dc.publisher.department.fl_str_mv | Escola de Veterinária e Zootecnia - EVZ (RG) |
| dc.publisher.initials.fl_str_mv | UFG |
| dc.publisher.none.fl_str_mv | Universidade Federal de Goiás |
| dc.publisher.program.fl_str_mv | Programa de Pós-graduação em Ciência Animal (EVZ) |
| dc.relation.cnpq.fl_str_mv | -7636512811479495338 |
| dc.relation.confidence.fl_str_mv | 600 600 600 |
| dc.relation.department.fl_str_mv | -6217552114249094582 |
| dc.relation.program.fl_str_mv | 4581960685150189167 |
| dc.rights.driver.fl_str_mv | http://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess |
| dc.source.none.fl_str_mv | reponame:Repositório Institucional da UFG instname:Universidade Federal de Goiás (UFG) instacron:UFG |
| dc.subject.cnpq.fl_str_mv | CIENCIA E TECNOLOGIA DE ALIMENTOS::TECNOLOGIA DE ALIMENTOS |
| dc.subject.por.fl_str_mv | Biopanning BoHV -1 Diagnóstico Epitopo descontínuo Glicoproteína B Diagnosis Discontinuous epitope Glycoprotein B |
| dc.title.alternative.eng.fl_str_mv | Prospecting and characterization of recombinant mimetic peptides to total antigens of herpesvirus type 1 by phage display |
| dc.title.por.fl_str_mv | Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display |
| dc.type.driver.fl_str_mv | info:eu-repo/semantics/doctoralThesis |
| dc.type.status.fl_str_mv | info:eu-repo/semantics/publishedVersion |
| description | Member of the Herpesviridae family, subfamily Alphaherpesvirinae, gender Varicellovirus, the bovine herpesvirus 1 (BoHV-1) has been associated with different clinical conditions (respiratory and genital/reproductive diseases) in cattle. There is no standard procedure to control or prevent infections caused by herpesviruses. In this sense, phage display was used to select new glycoprotein mimotopes antigen of BoHV-1 that has potential for use in vaccines and diagnostics. The phage display technique was performed using a linear random peptide library consisting of 12 amino acid residues fused to the protein III of M13 phage (no peptide) against BoHV-1 specific IgGs, purified by affinity chromatography. After three cycles of selection (biopanning) and amplification, 44 clones were isolated and their amino acid sequences were determined by sequencing generating 16 different sequences. ELISA, demonstrating the efficiency of selection from the specific antibodies, confirmed the reactivity of pooled clones. Another ELISA evaluated the individual specificity of the most frequent clones, the M13 phage was used as a negative control. We selected three peptides (B, C and E) with affinity for anti-BoHV-1 antibodies, and the E peptide (pepE), showed to have potential as antigen for antibody detection in a serological test for BoHV-1. Immunization of rabbits with the peptides induced specific production of serum antibodies, but they were not able do neutralize BoHV-1 cell lysis. The in silico analysis of the dodecapeptide E (1DRALYGPTVIDH12) enabled the identification of a new discontinuous epitope on the envelope glycoprotein B (gB Env) of bovine herpesvirus type 1 (BoHV-1). There is a short motif (338YKRD341) within a region of the env gB BoHV-1 with high similarity to motifs shared by dodecapeptide the N-terminal region (5YxARD1) of gB and HSV-1 (326YARD329), wherein the 328Arg residue is described as a target for neutralizing monoclonal antibodies (mAb) for HSV-1 gB. Besides the characterization of an antibody-binding site of the BoHV-1 Env gB, we have demonstrated that the phage-fused peptide has potential use as a reagent for virus diagnosis by phage-ELISA assay, discriminating BoHV-1 positive serum samples from negative ones. |
| eu_rights_str_mv | openAccess |
| format | doctoralThesis |
| id | UFG-2_b9aba9caa3cc628f1275a8fa67d775aa |
| identifier_str_mv | ALMEIDA, G. R. Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display. 2015. 60 f. Tese (Doutorado em Ciência Animal) - Universidade Federal de Goiás, Goiânia, 2015. |
| instacron_str | UFG |
| institution | UFG |
| instname_str | Universidade Federal de Goiás (UFG) |
| language | por |
| network_acronym_str | UFG-2 |
| network_name_str | Repositório Institucional da UFG |
| oai_identifier_str | oai:repositorio.bc.ufg.br:tede/5503 |
| publishDate | 2015 |
| publishDateSort | 2015 |
| publisher.none.fl_str_mv | Universidade Federal de Goiás |
| reponame_str | Repositório Institucional da UFG |
| repository.mail.fl_str_mv | grt.bc@ufg.br |
| repository.name.fl_str_mv | Repositório Institucional da UFG - Universidade Federal de Goiás (UFG) |
| repository_id_str | oai:repositorio.bc.ufg.br:tede/1234 |
| rights_invalid_str_mv | http://creativecommons.org/licenses/by/4.0/ |
| spelling | Souza, Guilherme Rocha Lino dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4700389E3Brito, Wilia Marta Elsner Diederichsen deBataus, Luiz Artur Mendeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4785339A7Souza, Guilherme Rocha Lino deFiaccadori, Fabiola SouzaKipnis, AndréCunha, Paulo Henrique Jorge daOliveira, Cairo Henrique Sousa dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4247180T2Almeida, Greyciele Rodrigues de2016-04-20T15:16:36Z2015-08-24ALMEIDA, G. R. Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display. 2015. 60 f. Tese (Doutorado em Ciência Animal) - Universidade Federal de Goiás, Goiânia, 2015.http://repositorio.bc.ufg.br/tede/handle/tede/5503Member of the Herpesviridae family, subfamily Alphaherpesvirinae, gender Varicellovirus, the bovine herpesvirus 1 (BoHV-1) has been associated with different clinical conditions (respiratory and genital/reproductive diseases) in cattle. There is no standard procedure to control or prevent infections caused by herpesviruses. In this sense, phage display was used to select new glycoprotein mimotopes antigen of BoHV-1 that has potential for use in vaccines and diagnostics. The phage display technique was performed using a linear random peptide library consisting of 12 amino acid residues fused to the protein III of M13 phage (no peptide) against BoHV-1 specific IgGs, purified by affinity chromatography. After three cycles of selection (biopanning) and amplification, 44 clones were isolated and their amino acid sequences were determined by sequencing generating 16 different sequences. ELISA, demonstrating the efficiency of selection from the specific antibodies, confirmed the reactivity of pooled clones. Another ELISA evaluated the individual specificity of the most frequent clones, the M13 phage was used as a negative control. We selected three peptides (B, C and E) with affinity for anti-BoHV-1 antibodies, and the E peptide (pepE), showed to have potential as antigen for antibody detection in a serological test for BoHV-1. Immunization of rabbits with the peptides induced specific production of serum antibodies, but they were not able do neutralize BoHV-1 cell lysis. The in silico analysis of the dodecapeptide E (1DRALYGPTVIDH12) enabled the identification of a new discontinuous epitope on the envelope glycoprotein B (gB Env) of bovine herpesvirus type 1 (BoHV-1). There is a short motif (338YKRD341) within a region of the env gB BoHV-1 with high similarity to motifs shared by dodecapeptide the N-terminal region (5YxARD1) of gB and HSV-1 (326YARD329), wherein the 328Arg residue is described as a target for neutralizing monoclonal antibodies (mAb) for HSV-1 gB. Besides the characterization of an antibody-binding site of the BoHV-1 Env gB, we have demonstrated that the phage-fused peptide has potential use as a reagent for virus diagnosis by phage-ELISA assay, discriminating BoHV-1 positive serum samples from negative ones.Membro da família Herpesviridae, subfamília Alphaherpesvirinae, gênero Varicellovirus, o herpesvírus bovino 1 (BoHV-1) tem sido associado a diferentes condições clínicas em bovinos (doenças respiratórias, genitais e falhas reprodutivas). Não existe um procedimento padrão para medidas de controle e profilaxia das infecções causadas por herpesvírus. Nesse sentido, o phage display foi utilizado com o objetivo de selecionar novos antígenos mimetopos de glicoproteínas do herpesvírus bovino 1 (BoHV-1) e que apresentam potencial para uso em vacinas e diagnóstico. A técnica de phage display foi realizada com a utilização de uma biblioteca de peptídeos randômicos e lineares composta de 12 resíduos de aminoácidos fusionada à pIII de fagos M13 (sem peptídeo), contra anticorpos anti-BoHV-1, purificados em coluna de cromatografia por afinidade. Após três ciclos de seleção (biopanning) e amplificação, 44 clones foram isolados e as sequências de aminoácidos dos peptídeos foram determinadas pelo sequenciamento gerando 16 sequencias diferentes. A reatividade do pool de clones foi confirmada por ELISA, demonstrando a eficiência da seleção a partir dos anticorpos específicos. Para avaliação da especificidade individual, realizou-se o ELISA dos clones mais frequentes, tendo como controle negativo o fago M13. Foram selecionados três peptídeos (B, C e E) com afinidade por anticorpos anti-BoHV-1, e um destes, o peptídeo E (pepE), apresentou potencial antigênico na detecção de anticorpos para o diagnóstico sorológico do BoHV-1. Nos testes de imunização em coelhos, os três peptídeos induziram a produção de anticorpos específicos, porém, estes não foram capazes de neutralizar a lise celular ocasionada pelo BoHV-1 em placa. A análise in silico do dodecapeptídeo E (1DRALYGPTVIDH12) possibilitou a identificação de um novo epitopo descontínuo na glicoproteína B de envelope (Env gB) do BoHV-1. Há um curto motivo (338YKRD341) dentro de uma região do gene Env gB do BoHV-1, com alta similaridade com os motivos compartilhados pelo dodecapeptídio da região N-terminal (5YxARD1) da gB e do Herpesvirus Humano 1 (HSV-1) (326YARD329), em que o resíduo 328Arg é descrito como um alvo para anticorpos monoclonais neutralizantes (mAb) para a gB do HSV-1. Concluindo, além da caracterização de um sítio de ligação ao anticorpo na Env gB do BoHV-1, o pepE expresso pelo fago tem potencial de utilização como reagente para o diagnóstico virológico por ensaio ELISA-fago, que discrimina amostras de soro positivas e negativas para o BoHV-1.application/pdfporUniversidade Federal de GoiásPrograma de Pós-graduação em Ciência Animal (EVZ)UFGBrasilEscola de Veterinária e Zootecnia - EVZ (RG)http://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessBiopanningBoHV -1DiagnósticoEpitopo descontínuoGlicoproteína BDiagnosisDiscontinuous epitopeGlycoprotein BCIENCIA E TECNOLOGIA DE ALIMENTOS::TECNOLOGIA DE ALIMENTOSProspecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage displayProspecting and characterization of recombinant mimetic peptides to total antigens of herpesvirus type 1 by phage displayinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis4581960685150189167600600600-6217552114249094582-7636512811479495338reponame:Repositório Institucional da UFGinstname:Universidade Federal de Goiás (UFG)instacron:UFGCC-LICENSElicense_urllicense_urltext/plain; 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| spellingShingle | Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display Almeida, Greyciele Rodrigues de Biopanning BoHV -1 Diagnóstico Epitopo descontínuo Glicoproteína B Diagnosis Discontinuous epitope Glycoprotein B CIENCIA E TECNOLOGIA DE ALIMENTOS::TECNOLOGIA DE ALIMENTOS |
| status_str | publishedVersion |
| title | Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display |
| title_full | Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display |
| title_fullStr | Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display |
| title_full_unstemmed | Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display |
| title_short | Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display |
| title_sort | Prospecção e caracterização de peptídeos recombinantes miméticos a antígenos totais de herpesvírus bovino 1 por meio de phage display |
| topic | Biopanning BoHV -1 Diagnóstico Epitopo descontínuo Glicoproteína B Diagnosis Discontinuous epitope Glycoprotein B CIENCIA E TECNOLOGIA DE ALIMENTOS::TECNOLOGIA DE ALIMENTOS |
| url | http://repositorio.bc.ufg.br/tede/handle/tede/5503 |