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DISSERTACAO 2008 Edelyncr Universidade de Brasília Programa de Pós-Graduação em Biologia Animal Construção da biblioteca de cDNA da glândula de peçonha do escorpião Opisthacanthus cayaporum e clonagem de genes que codificam para componentes da peçonha Édelyn Cristina Nunes Silva Orientadora: Profª Drª Elisabeth N. Ferroni Schwartz Dissertação apresentada ao Programa de Pós-Graduação em Biologia Animal da Universidade de Brasília como parte dos requisitos para a obtenção do título de Mestre. Brasília, 2008 1 UNIVERSIDADE DE BRASÍLIA INSTITUTO DE CIÊNCIAS BIOLÓGICAS PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA ANIMAL Dissertação de Mestrado Édelyn Cristina Nunes Silva Título: “Construção da biblioteca de cDNA da glândula de peçonha do escorpião Opisthacanthus cayaporum e clonagem de genes que codificam para componentes da peçonha” Comissão examinadora: Profa.Dra. Elisabeth N. Ferroni Schwartz Presidente/Orientadora CFS/UnB Prof. Dr. Luciano Paulino da Silva Profa. Dra. Ildinete Silva Pereira Embrapa/Cenargen CEL/UnB Profa. Dra. Márcia Renata Mortari Suplente, CFS/UnB Brasília, julho de 2008 2 DEDICATÓRIA A Deus Ao meu noivo Flávio A minha amada Mãe, Matilde A minha irmã Évelyn A Dra. Elisabeth Schwartz e ao Dr. Lourival Possani 3 AGRADECIMENTOS Esta dissertação de Mestrado só teve êxito porque ela é fruto do trabalho de muitas pessoas, de dois países, vários laboratórios, duas universidades... Assim tentarei agradecer a todos por essa vitória! Meus sinceros agradecimentos ao Prof. Dr. Lourival Possani do Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología da Universidad Autônoma do México, em Cuernavaca / Morelos, pela oportunidade de viajar ao México, conhecer esse maravilhoso país e trabalhar em seu renomado laboratório onde pude realizar grande parte dessa dissertação. Agradeço muitíssimo à minha orientadora Profa Dra Elisabeth Schwartz, por acreditar no meu trabalho, me dar a oportunidade de conhecimento, me auxiliar sempre em todas as etapas desse trabalho, por ser fonte de enorme conhecimento e trabalho. Sem ela esse trabalho nem sairia do papel. Ao laboratório de Biologia Molecular da Universidade de Brasília, às professoras Dras Andréa Maranhão, Ildinete Pereira, ao aluno de doutorado Marciano, por sempre me auxiliarem na obtenção dos dados para esse trabalho, por serem gentis e acessíveis para comigo. Agradeço ao Dr. Carlos Bloch Júnior e Dr. Luciano Paulino da Silva do Laboratório de Espectrometria de Massa, Embrapa/Cenargen, pela colaboração e inestimáveis ajudas. Ao professor Dr. Osmindo Júnior por sempre estar do meu lado, mesmo nos momentos de terrorismo. A sua esposa Janaína pelos “toques”, momentos de descontração e inestimável ajuda. Ao professor Dr. Carlos Alberto Schwartz pelas sempre oportunas dicas, pelo terrorismo que no fundo ajudava muito. Á professora Dra. Márcia Mortari pelas ótimas conversas profundas sobre profissão, pelos conhecimentos compartilhados, pelos momentos de descontração, por ser sempre estar disponível para uma palavra amiga. Obrigada também a sua ótima família: Gustavo e Luzitano. Como agradecer à Thalita e Naty? Seriam rolos de papel descrevendo a alegria que vocês me proporcionam, a base firme e otimista quando tudo tava dando errado, 4 companheiras nas horas divertidas e trágicas. Grandes amigas, esse trabalho tem um pedacinho de vocês. Agradeço ao admirável Jimmy Guerrero Vargas, que tem guerreiro não só no nome, que é exemplo de vida para mim, de força de vontade, responsabilidade e trabalho. Aos amigos do laboratório, Carol Barbosa, Carla Vizzoto, Solange, Poliana, Rafael Félix, Rafael Melani, Luís, Washigton, Cezinha, Leandro, Thompson, Professor Antônio Sebben, Flávio Dourado, Flávio Caixeta, pelos papos não-científicos, momentos de descontração e pela amizade nestes dois anos. Agradeço aos amigos mexicanos: Maria Juanita, Cynthia, Verna, Verônica, River, Blanca, Fred, Ligia Gonzáles, pelos dias maravilhosos que passei no México, pelo apoio no desenvolvimento desse trabalho, pela imensa ajuda no laboratório e pelos formidáveis passeios. Muchas gracias, ustedes están en mi corazón! A amiga Turca Figuem, minha companheira de quarto na casa da Yoli, agradeço por ser ótima companhia e por me mostrar o México que eu não conhecia. Aos amigos brasileiros Clara, Giu, Vanessa, Gabi por compreenderem minha ausência por uns tempos e serem ótimas companheiras. Ao meu amado noivo Flávio, por estar comigo em todos os momentos, por me auxiliar na escrita desse trabalho, por agüentar 40 dias sem mim, por me receber do México com todo o amor acumulado, por pesquisar na Internet sobre toxinas antimicrobianas, mesmo sem saber o que era isso.... E por transformar minha vida em um conto de fadas! Agradeço imensamente a minha querida Mamis, Matilde, por ser a base da minha vida, por ter investido em mim, por ser meu exemplo de luta e perseverança, pelo apoio no desenvolvimento desse Mestrado e por me amar sempre. Amo você Mamis! A minha querida irmã Évelyn, por me acordar pra fazer a dissertação, ser minha companheira por toda a vida, minha amiga incondicional... Agradeço ao Cnpq pelo financiamento e bolsa de estudos de Mestrado. Ao programa de Pós-graduação em Biologia Animal e ao Instituto de Biologia da Universidade de Brasília pelo apoio e oportunidade de trabalho. E mais importante, agradeço à Deus que me deu a dádiva da vida, rodeou ela de amigos e anjos-da-guarda e que permitiu a conquista de mais essa vitória. 5 SUMÁRIO INTRODUÇÃO.....................................................................................................................1 1. Escorpiões..........................................................................................................1 2. Composição da peçonha de escorpiões..............................................................2 2.1 Canal para Na+ voltagem-dependente e toxinas escorpiônicas moduladoras de canal para Na+ (NavTx)..............................................3 2.2 Canal para K+ e toxinas escorpiônicas bloqueadoras de canal para K+ ......................................................................................................... 5 2.3 Toxinas escorpiônicas moduladoras de canal para Ca2+ e moduladoras de canal para Cl-................................................................6 2.4 Toxinas sem pontes dissulfeto..........................................................7 Antimicrobianas.........................................................................7 Imunomoduladores.....................................................................8 Potencializadores de Bradicinina...............................................9 Hemolíticos................................................................................9 3. Estudos de proteômica e transcriptoma de peçonha de escorpiões....................9 4. Opisthacanthus cayaporum.............................................................................11 RELEVÂNCIA CIENTÍFICA...........................................................................................14 OBJETIVOS........................................................................................................................15 Gerais...............................................................................................................15 Específicos.......................................................................................................15 MATERIAIS E MÉTODOS...............................................................................................16 1. Soluções e Meios de cultivo.............................................................................16 2. Coleta e manutenção dos escorpiões................................................................16 3. Construção da Biblioteca de cDNA.................................................................16 3.1 Extração do RNA total..............................................................................16 3.2 Construção do Banco de cDNA...............................................................17 3.3 Ligação ao vetor pDNR-LIB (Clontech/UK)............................................20 4. Transformação bacteriana...............................................................................20 5. Seqüenciamento automático de DNA..............................................................21 6 6. Caracterização da Cayaporina..........................................................................22 5.1 Obtenção da Cayaporina...........................................................................23 5.2 Determinação da seqüência de aminoácidos ...........................................23 5.3 Atividade Biológica..................................................................................23 RESULTADOS....................................................................................................................25 1. Biblioteca de cDNA.........................................................................................25 2. Seqüência de nucleotídeos obtidos...................................................................25 Seqüências relacionadas a prováveis toxinas............................................29 Seqüências com cisteínas (cys) que formariam pontes dissulfeto..29 Prováveis fosfolipases....................................................................41 Seqüências sem cisteínas...............................................................44 Seqüências de nucleotídeos sem função atribuída.....................................50 3. Descrição do peptídeo Cayaporina...................................................................54
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