Obtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania major

Detalhes bibliográficos
Autor(a) principal: Freitas, Rodrigo de Barros
Data de Publicação: 2010
Tipo de documento: Dissertação
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/2423
Resumo: Leishmaniasis are parasitic diseases caused by protozoa of the Kinetoplastida order, Trypanossomatidae family and Leishmania genus. It currently represents a public health problem in Brazil and other countries in the world. The E-NTPDase or apyrases present in such parasites are mainly responsible for the degradation of nucleotides, especially extracellular di- and/or triphosphate, thus allowing the parasite to modulate an immune response in the host, which needs a purinergic signalling, and promoting the cellular development for its participation in the purine salvage pathway, in which the parasites are dependent. Monoclonal antibodies are originated of a hybridoma for the recognition of a specific antigen and can be selected for a desired specificity as well as produced in large scale and with good quality. This research aimed the expression of the heterologous L.major E-NTPDase-2 proteins and the production of monoclonal antibody-secreting hybridomas against such protein. A design of specific primers aiming the amplification of the coding region of the soluble portion (ecto-domain) of the E-NTPDase-2 was proposed, without the coding region for the amino-terminal peptide portion, resulting in a 1053-base-pair DNA. The amplicon was obtained by PCR amplification, cloned into an amplification (pJET-bunt) and expression (pET-21b) vector of bacterial system (E. coli DH5α). The protein expression of the ENTPDase- 2 was carried out in a bacterial system (E. coli BL-21) and the purification was performed by affinity chromatography on a Ni2+-agarose column (Ni-NTA). With the purified protein, immunizations of the mice were conducted with subsequent confirmation of the production of anti-NTPDase-2 antibodies, by means of DOT ELISA. The fusion of the B cells from the immunized mice, with the myeloma cells (Sp20), was performed in order to obtain the hybridoma. Then, hybridoma of L.major anti-E-NTPDase-2 were isolated making its usage possible in several future applications such as the diagnosis and treatment of canine leishmaniasis.
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spelling Freitas, Rodrigo de Barroshttp://lattes.cnpq.br/3286511632112171Salcedo, Joaquín Hernán Patarroyohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783313T4Lamêgo, Márcia Rogéria de Almeidahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782197P4Fietto, Juliana Lopes Rangelhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4790238D0Lima, Luciana Moreirahttp://lattes.cnpq.br/2013048264104100Afonso, Luis Carlos Croccohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4789739U0Bressan, Gustavo Costahttp://lattes.cnpq.br/11538532183477202015-03-26T13:07:30Z2011-10-062015-03-26T13:07:30Z2010-07-27FREITAS, Rodrigo de Barros. Obtainment of hybridomas for production of monoclonal antibodies against the E-NTPDase-2 of Leishmaniasis major protein. 2010. 95 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2010.http://locus.ufv.br/handle/123456789/2423Leishmaniasis are parasitic diseases caused by protozoa of the Kinetoplastida order, Trypanossomatidae family and Leishmania genus. It currently represents a public health problem in Brazil and other countries in the world. The E-NTPDase or apyrases present in such parasites are mainly responsible for the degradation of nucleotides, especially extracellular di- and/or triphosphate, thus allowing the parasite to modulate an immune response in the host, which needs a purinergic signalling, and promoting the cellular development for its participation in the purine salvage pathway, in which the parasites are dependent. Monoclonal antibodies are originated of a hybridoma for the recognition of a specific antigen and can be selected for a desired specificity as well as produced in large scale and with good quality. This research aimed the expression of the heterologous L.major E-NTPDase-2 proteins and the production of monoclonal antibody-secreting hybridomas against such protein. A design of specific primers aiming the amplification of the coding region of the soluble portion (ecto-domain) of the E-NTPDase-2 was proposed, without the coding region for the amino-terminal peptide portion, resulting in a 1053-base-pair DNA. The amplicon was obtained by PCR amplification, cloned into an amplification (pJET-bunt) and expression (pET-21b) vector of bacterial system (E. coli DH5α). The protein expression of the ENTPDase- 2 was carried out in a bacterial system (E. coli BL-21) and the purification was performed by affinity chromatography on a Ni2+-agarose column (Ni-NTA). With the purified protein, immunizations of the mice were conducted with subsequent confirmation of the production of anti-NTPDase-2 antibodies, by means of DOT ELISA. The fusion of the B cells from the immunized mice, with the myeloma cells (Sp20), was performed in order to obtain the hybridoma. Then, hybridoma of L.major anti-E-NTPDase-2 were isolated making its usage possible in several future applications such as the diagnosis and treatment of canine leishmaniasis.As Leishmanioses são doenças parasitárias causadas por protozoários da ordem Kinetoplastida, família Trypanossomatidae e gênero Leishmania que atualmente representam um problema de saúde pública no Brasil e em outros países do mundo. As E-NTPDases ou apirases, presentes nesses parasitos, têm como principal função a degradação de nucleotídeos, especialmente extracelulares di e/ou trifosfatados, permitindo ao parasito a modulação da resposta imune do hospedeiro, que necessecita da sinalização purinérgica, e favorecendo o desenvolvimento celular por sua participação na via de recuperação de purinas das quais os parasitos são dependentes. Os anticorpos monoclonais são oriundos de um hibridoma para reconhecimento de um determinado antígeno e podem ser selecionados para uma especificidade desejada, produzidos em grande quantidade e com boa qualidade. Este trabalho teve como objetivo a expressão heteróloga da proteína E-NTPDase-2 de L.major e produção de hibridomas secretores de anticorpos monoclonais contra esta proteína. Para isso foi feito o desenho de iniciadores específicos visando a amplificação da região codificante da porção solúvel (ecto-domínio) da E-NTPDase-2, sem a porção codificante para a região do peptídeo aminoterminal, perfazendo um DNA com 1053 pares de base. O amplicom foi obtido com amplificação por PCR, clonado em vetor de amplificação (pJET-bunt) e de expressão (pET-21b) em sistema bacteriano (E.coli DH5α). A expressão da proteína E-NTPDase-2 foi realizada em sistema bacteriano (E.coli BL-21) e a purificação foi feita por cromatografia de afinidade em coluna de Ni2+-agarose (Ni-NTA). Com a proteína purificada procedeu-se as imunizações dos camundongos e consequente confirmação da produção de anticorpos anti- NTPDase 2 por DOT ELISA. A fusão da células B dos camundongos imunizados, com as células de mieloma (Sp20) foi realizada para obteção dos hibridomas. Desta maneira foram isolados hibridomas anti-E-NTPDase-2 de L.major que poderão ser utilizados em várias aplicações futuras, como o diagnóstico e tratamento da Leishmaniose canina.Conselho Nacional de Desenvolvimento Científico e Tecnológicoapplication/pdfporUniversidade Federal de ViçosaMestrado em Bioquímica AgrícolaUFVBRBioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animalHibridomasE-NTPDase-2Leishmania majorHybridomasE-NTPDase-2Leishmania majorCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARObtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania majorObtainment of hybridomas for production of monoclonal antibodies against the E-NTPDase-2 of Leishmaniasis major proteininfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf2695751https://locus.ufv.br//bitstream/123456789/2423/1/texto%20completo.pdfd0202b62ed6a3aaeac0ae817f4c6684eMD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain163318https://locus.ufv.br//bitstream/123456789/2423/2/texto%20completo.pdf.txtd976c41de983f0a21a28f74ac2bbca72MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3547https://locus.ufv.br//bitstream/123456789/2423/3/texto%20completo.pdf.jpg8d2485ce5c8e59e0380fbfdf15c77d71MD53123456789/24232016-04-08 23:02:32.828oai:locus.ufv.br:123456789/2423Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-09T02:02:32LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv Obtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania major
dc.title.alternative.eng.fl_str_mv Obtainment of hybridomas for production of monoclonal antibodies against the E-NTPDase-2 of Leishmaniasis major protein
title Obtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania major
spellingShingle Obtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania major
Freitas, Rodrigo de Barros
Hibridomas
E-NTPDase-2
Leishmania major
Hybridomas
E-NTPDase-2
Leishmania major
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
title_short Obtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania major
title_full Obtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania major
title_fullStr Obtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania major
title_full_unstemmed Obtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania major
title_sort Obtenção de hibridomas para a produção de anticorpos monoclonais contra a proteína E-NTPDase-2 de Leishmania major
author Freitas, Rodrigo de Barros
author_facet Freitas, Rodrigo de Barros
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://lattes.cnpq.br/3286511632112171
dc.contributor.author.fl_str_mv Freitas, Rodrigo de Barros
dc.contributor.advisor-co1.fl_str_mv Salcedo, Joaquín Hernán Patarroyo
dc.contributor.advisor-co1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783313T4
dc.contributor.advisor-co2.fl_str_mv Lamêgo, Márcia Rogéria de Almeida
dc.contributor.advisor-co2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782197P4
dc.contributor.advisor1.fl_str_mv Fietto, Juliana Lopes Rangel
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4790238D0
dc.contributor.referee1.fl_str_mv Lima, Luciana Moreira
dc.contributor.referee1Lattes.fl_str_mv http://lattes.cnpq.br/2013048264104100
dc.contributor.referee2.fl_str_mv Afonso, Luis Carlos Crocco
dc.contributor.referee2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4789739U0
dc.contributor.referee3.fl_str_mv Bressan, Gustavo Costa
dc.contributor.referee3Lattes.fl_str_mv http://lattes.cnpq.br/1153853218347720
contributor_str_mv Salcedo, Joaquín Hernán Patarroyo
Lamêgo, Márcia Rogéria de Almeida
Fietto, Juliana Lopes Rangel
Lima, Luciana Moreira
Afonso, Luis Carlos Crocco
Bressan, Gustavo Costa
dc.subject.por.fl_str_mv Hibridomas
E-NTPDase-2
Leishmania major
topic Hibridomas
E-NTPDase-2
Leishmania major
Hybridomas
E-NTPDase-2
Leishmania major
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
dc.subject.eng.fl_str_mv Hybridomas
E-NTPDase-2
Leishmania major
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
description Leishmaniasis are parasitic diseases caused by protozoa of the Kinetoplastida order, Trypanossomatidae family and Leishmania genus. It currently represents a public health problem in Brazil and other countries in the world. The E-NTPDase or apyrases present in such parasites are mainly responsible for the degradation of nucleotides, especially extracellular di- and/or triphosphate, thus allowing the parasite to modulate an immune response in the host, which needs a purinergic signalling, and promoting the cellular development for its participation in the purine salvage pathway, in which the parasites are dependent. Monoclonal antibodies are originated of a hybridoma for the recognition of a specific antigen and can be selected for a desired specificity as well as produced in large scale and with good quality. This research aimed the expression of the heterologous L.major E-NTPDase-2 proteins and the production of monoclonal antibody-secreting hybridomas against such protein. A design of specific primers aiming the amplification of the coding region of the soluble portion (ecto-domain) of the E-NTPDase-2 was proposed, without the coding region for the amino-terminal peptide portion, resulting in a 1053-base-pair DNA. The amplicon was obtained by PCR amplification, cloned into an amplification (pJET-bunt) and expression (pET-21b) vector of bacterial system (E. coli DH5α). The protein expression of the ENTPDase- 2 was carried out in a bacterial system (E. coli BL-21) and the purification was performed by affinity chromatography on a Ni2+-agarose column (Ni-NTA). With the purified protein, immunizations of the mice were conducted with subsequent confirmation of the production of anti-NTPDase-2 antibodies, by means of DOT ELISA. The fusion of the B cells from the immunized mice, with the myeloma cells (Sp20), was performed in order to obtain the hybridoma. Then, hybridoma of L.major anti-E-NTPDase-2 were isolated making its usage possible in several future applications such as the diagnosis and treatment of canine leishmaniasis.
publishDate 2010
dc.date.issued.fl_str_mv 2010-07-27
dc.date.available.fl_str_mv 2011-10-06
2015-03-26T13:07:30Z
dc.date.accessioned.fl_str_mv 2015-03-26T13:07:30Z
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dc.identifier.citation.fl_str_mv FREITAS, Rodrigo de Barros. Obtainment of hybridomas for production of monoclonal antibodies against the E-NTPDase-2 of Leishmaniasis major protein. 2010. 95 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2010.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/2423
identifier_str_mv FREITAS, Rodrigo de Barros. Obtainment of hybridomas for production of monoclonal antibodies against the E-NTPDase-2 of Leishmaniasis major protein. 2010. 95 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2010.
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