Expressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongos
Autor(a) principal: | |
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Data de Publicação: | 2011 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFPel - Guaiaca |
Texto Completo: | http://repositorio.ufpel.edu.br/handle/ri/1296 |
Resumo: | Several recombinant proteins have been studied as immunocontraception agents in mammals, among them the Gonadotropin-releasing hormone (GnRH). Nevertheless, due to its low molecular weight, GnRH needs to be conjugated with a carrier protein. Among the eukaryotic platforms the yeast Pichia pastoris, which are able of promoting post-translational modifications to expressed recombinant protein. The aim of this study was to clone and express the GnRH/LTB chimera in P. pastoris, and to test it as an immunocontraceptive. In order to accomplish this, sequences of P. pastoris codons usage were synthesized and cloned into the plasmid pPICZαB. The plasmid containing the sequence were propagated and transformed into yeast by electroporation. The transformed colonies were selected by resistance to antibiotics and confirmed by Colony Blotting. The selected clone was cultivated, induced and the protein expressed in supernatant. The protein was concentrated and used as a vaccine antigen. BALB/c males mice were vaccinated twice 15 apart with 100µg de LTB/GnRH adjuvanted with aluminum hydroxide. The immune response was evaluated by ELISA, showing seraconversion in vaccinated animals. The sperm concentration in the control group was 16.93 x 107 and 8.00 x 107 sperm/mL in the immunized group. Vaccination caused a reduction of spermatogenesis (p < 0,05), vacuolization and disorganization of the seminiferous tubules. We concluded that the yeast was able to express the recombinant protein LTB/GnRH, and it demonstrated immunocontraceptive potential in mice. |
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2014-08-20T13:32:59Z2011-09-142014-08-20T13:32:59Z2011-07-18PEREIRA, Juliano Lacava. Heterologous expression of chimera synthetic LTB/GnRH in Pichia pastoris and its effect on immune response and in the seminiferous epithelium of mice. 2011. 62 f. Dissertação (Mestrado em Biotecnologia) - Universidade Federal de Pelotas, Pelotas, 2011.http://repositorio.ufpel.edu.br/handle/ri/1296Several recombinant proteins have been studied as immunocontraception agents in mammals, among them the Gonadotropin-releasing hormone (GnRH). Nevertheless, due to its low molecular weight, GnRH needs to be conjugated with a carrier protein. Among the eukaryotic platforms the yeast Pichia pastoris, which are able of promoting post-translational modifications to expressed recombinant protein. The aim of this study was to clone and express the GnRH/LTB chimera in P. pastoris, and to test it as an immunocontraceptive. In order to accomplish this, sequences of P. pastoris codons usage were synthesized and cloned into the plasmid pPICZαB. The plasmid containing the sequence were propagated and transformed into yeast by electroporation. The transformed colonies were selected by resistance to antibiotics and confirmed by Colony Blotting. The selected clone was cultivated, induced and the protein expressed in supernatant. The protein was concentrated and used as a vaccine antigen. BALB/c males mice were vaccinated twice 15 apart with 100µg de LTB/GnRH adjuvanted with aluminum hydroxide. The immune response was evaluated by ELISA, showing seraconversion in vaccinated animals. The sperm concentration in the control group was 16.93 x 107 and 8.00 x 107 sperm/mL in the immunized group. Vaccination caused a reduction of spermatogenesis (p < 0,05), vacuolization and disorganization of the seminiferous tubules. We concluded that the yeast was able to express the recombinant protein LTB/GnRH, and it demonstrated immunocontraceptive potential in mice.Diversas proteínas recombinantes estão sendo pesquisadas para utilização na imunocontracepção de mamíferos. Entre elas, o hormônio liberador de gonadotrofinas (GnRH), entretanto devido ao seu baixo peso molecular há necessidade de associá-lo a uma molécula carreadora. Dentre as plataformas eucarióticas, encontra-se a levedura Pichia pastoris que tem possibilitado expressão e modificações pós-traducionais das proteínas recombinantes. O objetivo deste trabalho foi clonar e expressar a quimera LTB/GnRH em P. pastoris, visando sua utilização como imunocontraceptivo. Para tanto, sequências com os códons preferenciais de P. pastoris da proteína foram sintetizadas e clonada no plasmídeo pPICZαB. O plasmídeo foi propagado e transformado na levedura por eletroporação. As colônias transformadas foram selecionadas por resistência antibiótica e confirmadas por Colony Blotting. O clone selecionado foi cultivado e após indução expressou a proteína no sobrenadante. A proteína foi concentrada e utilizada como antígeno vacinal. Camundongos BALB/c machos foram vacinados com duas doses de 100µg de LTB/GnRH adsorvidas em hidróxido de alumínio com intervalo de 15 dias. A resposta imune foi avaliada por ELISA, mostrando soroconversão nos animais imunizados. A concentração espermática no grupo controle foi 16,93 x 107 e no grupo imunizado 8,00 x 107 espermatozóides/mL. A imunização induziu a redução da espermatogênese (p < 0,05), vacuolização nos túbulos seminíferos e desorganização testicular. Concluiu-se que a levedura foi capaz de expressar a proteína LTB/GnRH recombinante e a mesma apresentou potencial imunocontraceptivo em camundongos.application/pdfporUniversidade Federal de PelotasPrograma de Pós-Graduação em BiotecnologiaUFPelBRBiotecnologiaImunocontraceptivoVacinas recombinantesEspermatogêneseLeveduraImmunocontraceptiveRecombinants vaccinesSpermatogenesisYeastCNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIA::IMUNOGENETICAExpressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongosHeterologous expression of chimera synthetic LTB/GnRH in Pichia pastoris and its effect on immune response and in the seminiferous epithelium of miceinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesishttp://lattes.cnpq.br/6037031116674928http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763076J5Leite, Fabio Pereira LeivasPereira, Juliano Lacavainfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFPel - Guaiacainstname:Universidade Federal de Pelotas (UFPEL)instacron:UFPELORIGINALdissertacao_juliano_lacava_pereira.pdfapplication/pdf1194037http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1296/1/dissertacao_juliano_lacava_pereira.pdf9990bc185909cc08288cd6230c0a9be6MD51open accessTEXTdissertacao_juliano_lacava_pereira.pdf.txtdissertacao_juliano_lacava_pereira.pdf.txtExtracted Texttext/plain103715http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1296/2/dissertacao_juliano_lacava_pereira.pdf.txt7a1b087652551bf18f535c1188415bbbMD52open accessTHUMBNAILdissertacao_juliano_lacava_pereira.pdf.jpgdissertacao_juliano_lacava_pereira.pdf.jpgGenerated Thumbnailimage/jpeg1761http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/1296/3/dissertacao_juliano_lacava_pereira.pdf.jpg3a662519b78819d33f2843efc2640834MD53open access123456789/12962019-08-23 09:16:41.737open accessoai:guaiaca.ufpel.edu.br:123456789/1296Repositório InstitucionalPUBhttp://repositorio.ufpel.edu.br/oai/requestrippel@ufpel.edu.br || repositorio@ufpel.edu.br || aline.batista@ufpel.edu.bropendoar:2019-08-23T12:16:41Repositório Institucional da UFPel - Guaiaca - Universidade Federal de Pelotas (UFPEL)false |
dc.title.por.fl_str_mv |
Expressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongos |
dc.title.alternative.eng.fl_str_mv |
Heterologous expression of chimera synthetic LTB/GnRH in Pichia pastoris and its effect on immune response and in the seminiferous epithelium of mice |
title |
Expressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongos |
spellingShingle |
Expressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongos Pereira, Juliano Lacava Imunocontraceptivo Vacinas recombinantes Espermatogênese Levedura Immunocontraceptive Recombinants vaccines Spermatogenesis Yeast CNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIA::IMUNOGENETICA |
title_short |
Expressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongos |
title_full |
Expressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongos |
title_fullStr |
Expressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongos |
title_full_unstemmed |
Expressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongos |
title_sort |
Expressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongos |
author |
Pereira, Juliano Lacava |
author_facet |
Pereira, Juliano Lacava |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/6037031116674928 |
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 |
Pereira, Juliano Lacava |
contributor_str_mv |
Leite, Fabio Pereira Leivas |
dc.subject.por.fl_str_mv |
Imunocontraceptivo Vacinas recombinantes Espermatogênese Levedura |
topic |
Imunocontraceptivo Vacinas recombinantes Espermatogênese Levedura Immunocontraceptive Recombinants vaccines Spermatogenesis Yeast CNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIA::IMUNOGENETICA |
dc.subject.eng.fl_str_mv |
Immunocontraceptive Recombinants vaccines Spermatogenesis Yeast |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIA::IMUNOGENETICA |
description |
Several recombinant proteins have been studied as immunocontraception agents in mammals, among them the Gonadotropin-releasing hormone (GnRH). Nevertheless, due to its low molecular weight, GnRH needs to be conjugated with a carrier protein. Among the eukaryotic platforms the yeast Pichia pastoris, which are able of promoting post-translational modifications to expressed recombinant protein. The aim of this study was to clone and express the GnRH/LTB chimera in P. pastoris, and to test it as an immunocontraceptive. In order to accomplish this, sequences of P. pastoris codons usage were synthesized and cloned into the plasmid pPICZαB. The plasmid containing the sequence were propagated and transformed into yeast by electroporation. The transformed colonies were selected by resistance to antibiotics and confirmed by Colony Blotting. The selected clone was cultivated, induced and the protein expressed in supernatant. The protein was concentrated and used as a vaccine antigen. BALB/c males mice were vaccinated twice 15 apart with 100µg de LTB/GnRH adjuvanted with aluminum hydroxide. The immune response was evaluated by ELISA, showing seraconversion in vaccinated animals. The sperm concentration in the control group was 16.93 x 107 and 8.00 x 107 sperm/mL in the immunized group. Vaccination caused a reduction of spermatogenesis (p < 0,05), vacuolization and disorganization of the seminiferous tubules. We concluded that the yeast was able to express the recombinant protein LTB/GnRH, and it demonstrated immunocontraceptive potential in mice. |
publishDate |
2011 |
dc.date.available.fl_str_mv |
2011-09-14 2014-08-20T13:32:59Z |
dc.date.issued.fl_str_mv |
2011-07-18 |
dc.date.accessioned.fl_str_mv |
2014-08-20T13:32:59Z |
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 |
PEREIRA, Juliano Lacava. Heterologous expression of chimera synthetic LTB/GnRH in Pichia pastoris and its effect on immune response and in the seminiferous epithelium of mice. 2011. 62 f. Dissertação (Mestrado em Biotecnologia) - Universidade Federal de Pelotas, Pelotas, 2011. |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufpel.edu.br/handle/ri/1296 |
identifier_str_mv |
PEREIRA, Juliano Lacava. Heterologous expression of chimera synthetic LTB/GnRH in Pichia pastoris and its effect on immune response and in the seminiferous epithelium of mice. 2011. 62 f. Dissertação (Mestrado em Biotecnologia) - Universidade Federal de Pelotas, Pelotas, 2011. |
url |
http://repositorio.ufpel.edu.br/handle/ri/1296 |
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por |
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por |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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Universidade Federal de Pelotas |
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Programa de Pós-Graduação em Biotecnologia |
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UFPel |
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BR |
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Biotecnologia |
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Universidade Federal de Pelotas |
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