Expressão heteróloga da quimera LTB/GnRH sintética em Pichia pastoris e seu efeito na resposta imunológica e no epitélio seminífero de camundongos

Detalhes bibliográficos
Autor(a) principal: Pereira, Juliano Lacava
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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spelling 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
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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.
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