Expressão do receptor de vitamina D recombinante: um importante alvo biológico
Ano de defesa: | 2015 |
---|---|
Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | |
Tipo de documento: | Dissertação |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade Estadual de Feira de Santana
|
Programa de Pós-Graduação: |
Mestrado Acadêmico em Biotecnologia
|
Departamento: |
DEPARTAMENTO DE CIÊNCIAS BIOLÓGICAS
|
País: |
Brasil
|
Palavras-chave em Português: | |
Palavras-chave em Inglês: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://localhost:8080/tede/handle/tede/209 |
Resumo: | The vitamin D receptor (VDR) is a cytoplasmic transcription factor and when activated by its ligand, translocates to the nucleus and interacts with DNA in the promoter regions with which has affinity, acting as a modulator of gene transcription and thereby producing multiple biological effects. Its main activities are the regulation and maintenance of plasma levels of calcium and phosphorus, as well as presenting immunomodulatory activities, such as the suppression of T cell activation, formation of secretion patterns of cytokines, modulation of proliferation and interference in the apoptosis. So this receptor is an important target for drugs that can help in the discovery of new immunomodulators. The present work had the objective to produce the recombinant vitamin D receptor in its soluble form for conducting future assays of drug screening of potential immunomodulators and drug-receptor interaction studies. For this, we used initially Escherichia coli expression system transformed with the plasmid HS_VDR_EC1-PQE T7, but it was only possible to obtain the protein in the insoluble fraction, even varying the temperature, time of induction and IPTG concentration. In an attempt to obtain soluble VDR, we used a eukaryotic expression system in insect cells using the baculovirus as a vector. It was built a vector, pFASTBacHT_VDR, which had the sequence of this protein cloned from pCMX.VDR. From there, it was possible to obtain recombinant bacmids used in transfection of insect cells, generating recombinant baculovirus, to then proceed with the expression of VDR. |
id |
UEFS_253c6cf79ae06939fcfa4bd16f540227 |
---|---|
oai_identifier_str |
oai:tede2.uefs.br:8080:tede/209 |
network_acronym_str |
UEFS |
network_name_str |
Biblioteca Digital de Teses e Dissertações da UEFS |
repository_id_str |
|
spelling |
Soares, Milena Botelho Pereirahttp://lattes.cnpq.br/9412047478182269Thomaz, Aline Machado2015-09-21T13:18:11Z2015-09-27THOMAZ, Aline machado. Expressão do receptor de vitamina D recombinante: um importante alvo biológico. 2015. 79f. Dissertação (Mestrado Acadêmico em Biotecnologia)- Universidade Estadual de Feira de Santana, Feira de Santana, 2013.http://localhost:8080/tede/handle/tede/209The vitamin D receptor (VDR) is a cytoplasmic transcription factor and when activated by its ligand, translocates to the nucleus and interacts with DNA in the promoter regions with which has affinity, acting as a modulator of gene transcription and thereby producing multiple biological effects. Its main activities are the regulation and maintenance of plasma levels of calcium and phosphorus, as well as presenting immunomodulatory activities, such as the suppression of T cell activation, formation of secretion patterns of cytokines, modulation of proliferation and interference in the apoptosis. So this receptor is an important target for drugs that can help in the discovery of new immunomodulators. The present work had the objective to produce the recombinant vitamin D receptor in its soluble form for conducting future assays of drug screening of potential immunomodulators and drug-receptor interaction studies. For this, we used initially Escherichia coli expression system transformed with the plasmid HS_VDR_EC1-PQE T7, but it was only possible to obtain the protein in the insoluble fraction, even varying the temperature, time of induction and IPTG concentration. In an attempt to obtain soluble VDR, we used a eukaryotic expression system in insect cells using the baculovirus as a vector. It was built a vector, pFASTBacHT_VDR, which had the sequence of this protein cloned from pCMX.VDR. From there, it was possible to obtain recombinant bacmids used in transfection of insect cells, generating recombinant baculovirus, to then proceed with the expression of VDR.O receptor de vitamina D (VDR) é um fator de transcrição gênica citoplasmático e, quando ativado pelo seu ligante, transloca-se para o núcleo e interage com regiões promotoras no DNA com a qual apresenta afinidade, atuando como fator modulador da transcrição gênica e assim produzindo múltiplos efeitos biológicos. Suas principais atividades são a regulação e a manutenção dos níveis plasmáticos de cálcio e fósforo, e apresenta atividades imunomoduladoras, como a supressão da ativação de células T, formação de padrões de secreção de citocinas, a modulação da proliferação e interferência na apoptose. Sendo assim, esse receptor representa um importante alvo de drogas que pode contribuir na descoberta de novos fármacos com ação imunomoduladora. O presente trabalho teve, como objetivo, produzir o VDR recombinante na forma solúvel para a realização de futuros ensaios de triagem de potenciais drogas com ação imunomoduladora e estudos de interação droga-receptor. Para isso, foi utilizado, inicialmente, o sistema de expressão em Escherichia coli, utilizando o plasmídeo HS_VDR_EC1-PQE T7, porém só foi possível obter a proteína na fração insolúvel, mesmo variando a temperatura, o tempo de indução e a concentração de IPTG. Na tentativa de obter o VDR solúvel, foi utilizado um sistema de expressão eucariótico em células de inseto, utilizando como vetor o baculovírus. Foi construído um vetor pFASTBacHT_VDR, o qual teve a sequência desta proteína clonada a partir do pCMX.VDR. A partir daí, foi possível obter bacmídeos recombinantes, utilizados na transfecção de células de inseto, gerando baculovírus recombinantes, para, então, seguir com a expressão do VDR.Submitted by Verena Bastos (verena@uefs.br) on 2015-09-21T13:18:11Z No. of bitstreams: 1 DISSERTACAO FINAL ALINE THOMAZ.pdf: 1680438 bytes, checksum: 85bf2d7886a394df745618b6fd50d435 (MD5)Made available in DSpace on 2015-09-21T13:18:11Z (GMT). No. of bitstreams: 1 DISSERTACAO FINAL ALINE THOMAZ.pdf: 1680438 bytes, checksum: 85bf2d7886a394df745618b6fd50d435 (MD5) Previous issue date: 2015-09-27Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESapplication/pdfporUniversidade Estadual de Feira de SantanaMestrado Acadêmico em BiotecnologiaUEFSBrasilDEPARTAMENTO DE CIÊNCIAS BIOLÓGICASReceptor de vitamina DProteína recombinanteEscherichia coliBaculovírusVitamin D receptorRecombinant proteinEscherichia coliBaculovirusCIENCIAS BIOLOGICASExpressão do receptor de vitamina D recombinante: um importante alvo biológicoinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis7701973706309601282600600600600-6971480722008537872-34391788430682021612075167498588264571info:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UEFSinstname:Universidade Estadual de Feira de Santana (UEFS)instacron:UEFSORIGINALDISSERTACAO FINAL ALINE THOMAZ.pdfDISSERTACAO FINAL ALINE THOMAZ.pdfapplication/pdf1680438http://tede2.uefs.br:8080/bitstream/tede/209/2/DISSERTACAO+FINAL+ALINE+THOMAZ.pdf85bf2d7886a394df745618b6fd50d435MD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82165http://tede2.uefs.br:8080/bitstream/tede/209/1/license.txtbd3efa91386c1718a7f26a329fdcb468MD51tede/2092015-09-21 10:18:11.925oai:tede2.uefs.br:8080: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Biblioteca Digital de Teses e Dissertaçõeshttp://tede2.uefs.br:8080/PUBhttp://tede2.uefs.br:8080/oai/requestbcuefs@uefs.br|| bcref@uefs.br||bcuefs@uefs.bropendoar:2015-09-21T13:18:11Biblioteca Digital de Teses e Dissertações da UEFS - Universidade Estadual de Feira de Santana (UEFS)false |
dc.title.por.fl_str_mv |
Expressão do receptor de vitamina D recombinante: um importante alvo biológico |
title |
Expressão do receptor de vitamina D recombinante: um importante alvo biológico |
spellingShingle |
Expressão do receptor de vitamina D recombinante: um importante alvo biológico Thomaz, Aline Machado Receptor de vitamina D Proteína recombinante Escherichia coli Baculovírus Vitamin D receptor Recombinant protein Escherichia coli Baculovirus CIENCIAS BIOLOGICAS |
title_short |
Expressão do receptor de vitamina D recombinante: um importante alvo biológico |
title_full |
Expressão do receptor de vitamina D recombinante: um importante alvo biológico |
title_fullStr |
Expressão do receptor de vitamina D recombinante: um importante alvo biológico |
title_full_unstemmed |
Expressão do receptor de vitamina D recombinante: um importante alvo biológico |
title_sort |
Expressão do receptor de vitamina D recombinante: um importante alvo biológico |
author |
Thomaz, Aline Machado |
author_facet |
Thomaz, Aline Machado |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Soares, Milena Botelho Pereira |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/9412047478182269 |
dc.contributor.author.fl_str_mv |
Thomaz, Aline Machado |
contributor_str_mv |
Soares, Milena Botelho Pereira |
dc.subject.por.fl_str_mv |
Receptor de vitamina D Proteína recombinante Escherichia coli Baculovírus |
topic |
Receptor de vitamina D Proteína recombinante Escherichia coli Baculovírus Vitamin D receptor Recombinant protein Escherichia coli Baculovirus CIENCIAS BIOLOGICAS |
dc.subject.eng.fl_str_mv |
Vitamin D receptor Recombinant protein Escherichia coli Baculovirus |
dc.subject.cnpq.fl_str_mv |
CIENCIAS BIOLOGICAS |
description |
The vitamin D receptor (VDR) is a cytoplasmic transcription factor and when activated by its ligand, translocates to the nucleus and interacts with DNA in the promoter regions with which has affinity, acting as a modulator of gene transcription and thereby producing multiple biological effects. Its main activities are the regulation and maintenance of plasma levels of calcium and phosphorus, as well as presenting immunomodulatory activities, such as the suppression of T cell activation, formation of secretion patterns of cytokines, modulation of proliferation and interference in the apoptosis. So this receptor is an important target for drugs that can help in the discovery of new immunomodulators. The present work had the objective to produce the recombinant vitamin D receptor in its soluble form for conducting future assays of drug screening of potential immunomodulators and drug-receptor interaction studies. For this, we used initially Escherichia coli expression system transformed with the plasmid HS_VDR_EC1-PQE T7, but it was only possible to obtain the protein in the insoluble fraction, even varying the temperature, time of induction and IPTG concentration. In an attempt to obtain soluble VDR, we used a eukaryotic expression system in insect cells using the baculovirus as a vector. It was built a vector, pFASTBacHT_VDR, which had the sequence of this protein cloned from pCMX.VDR. From there, it was possible to obtain recombinant bacmids used in transfection of insect cells, generating recombinant baculovirus, to then proceed with the expression of VDR. |
publishDate |
2015 |
dc.date.accessioned.fl_str_mv |
2015-09-21T13:18:11Z |
dc.date.issued.fl_str_mv |
2015-09-27 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
THOMAZ, Aline machado. Expressão do receptor de vitamina D recombinante: um importante alvo biológico. 2015. 79f. Dissertação (Mestrado Acadêmico em Biotecnologia)- Universidade Estadual de Feira de Santana, Feira de Santana, 2013. |
dc.identifier.uri.fl_str_mv |
http://localhost:8080/tede/handle/tede/209 |
identifier_str_mv |
THOMAZ, Aline machado. Expressão do receptor de vitamina D recombinante: um importante alvo biológico. 2015. 79f. Dissertação (Mestrado Acadêmico em Biotecnologia)- Universidade Estadual de Feira de Santana, Feira de Santana, 2013. |
url |
http://localhost:8080/tede/handle/tede/209 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.program.fl_str_mv |
7701973706309601282 |
dc.relation.confidence.fl_str_mv |
600 600 600 600 |
dc.relation.department.fl_str_mv |
-6971480722008537872 |
dc.relation.cnpq.fl_str_mv |
-3439178843068202161 |
dc.relation.sponsorship.fl_str_mv |
2075167498588264571 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Estadual de Feira de Santana |
dc.publisher.program.fl_str_mv |
Mestrado Acadêmico em Biotecnologia |
dc.publisher.initials.fl_str_mv |
UEFS |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
DEPARTAMENTO DE CIÊNCIAS BIOLÓGICAS |
publisher.none.fl_str_mv |
Universidade Estadual de Feira de Santana |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da UEFS instname:Universidade Estadual de Feira de Santana (UEFS) instacron:UEFS |
instname_str |
Universidade Estadual de Feira de Santana (UEFS) |
instacron_str |
UEFS |
institution |
UEFS |
reponame_str |
Biblioteca Digital de Teses e Dissertações da UEFS |
collection |
Biblioteca Digital de Teses e Dissertações da UEFS |
bitstream.url.fl_str_mv |
http://tede2.uefs.br:8080/bitstream/tede/209/2/DISSERTACAO+FINAL+ALINE+THOMAZ.pdf http://tede2.uefs.br:8080/bitstream/tede/209/1/license.txt |
bitstream.checksum.fl_str_mv |
85bf2d7886a394df745618b6fd50d435 bd3efa91386c1718a7f26a329fdcb468 |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 |
repository.name.fl_str_mv |
Biblioteca Digital de Teses e Dissertações da UEFS - Universidade Estadual de Feira de Santana (UEFS) |
repository.mail.fl_str_mv |
bcuefs@uefs.br|| bcref@uefs.br||bcuefs@uefs.br |
_version_ |
1800214647811342336 |