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Anderson Oliveira Do Carmo Busca Por Novos Antígenos Para a Produção De Soro Antiescorpiônico

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Anderson Oliveira Do Carmo Busca Por Novos Antígenos Para a Produção De Soro Antiescorpiônico Anderson Oliveira do Carmo Busca por novos antígenos para a produção de soro antiescorpiônico: Construção de antígenos quiméricos e caracterização de proteases da peçonha de T. serrulatus. Tese apresentada ao curso de pós-graduação em Genética do Instituto de Ciências Biológicas da Universidade Federal de Minas Gerais com requisito parcial para obtenção de título de Doutor em Genética. Orientador: Evanguedes Kalapothakis Universidade Federal de Minas Gerais Instituto de Ciências Biológicas Departamento de Biologia Geral Programa de Pós-graduação em Genética Belo Horizonte, 2015. 043 Carmo, Anderson Oliveira do. Busca por novos antígenos para a produção de soro antiescorpiônico: construção de antígenos quiméricos e caracterização de proteases da peçonha de T. serrulatus [manuscrito] / Anderson Oliveira do Carmo. – 2015. 157 f. : il. ; 29,5 cm. Orientador: Evanguedes Kalapothakis. Tese (doutorado) – Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas. 1. Tityus serrulatus - Teses. 2. Escorpião – Teses. 3. Proteína quimera. 4. Protease. 5. Anticorpos. 6. Anti-veneno. 7. Genética – Teses. I. Kalapothakis, Evanguedes. II. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. III. Título. CDU: 575 Este trabalho foi realizado com o apoio das seguintes instituições: - Departamento de Biologia Geral – ICB – UFMG; - Departamento de Bioquímica e Imunologia – ICB – UFMG; - Fundação de Amparo a Pesquisa de Minas Gerais – FAPEMIG; - Conselho Nacional de Pesquisa – CNPq; - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES. VI Dedico esse trabalho a minha família. VII Agradecimentos Gostaria de agradecer ao programa de Pós Graduação em Genética e ao departamento de Biologia Geral pela oportunidade e apoio para o desenvolvimento desse trabalho e pela oportunidade de aprendizado e convívio com grandes pesquisadores do Brasil. As agências de fomento CAPES, CNPq e FAPEMIG pelo financiamento desse projeto e pela bolsa de doutorado. Ao professor Evanguedes Kalapothakis pelo apoio total durante todo o tempo de vivência. Você é um ótimo profissional no qual me espelho, com grande competência, sensibilidade e experiência, que sempre que necessário me indicou a direção certa a seguir para superar as adversidades surgidas durante esse trabalho. A banca por ter aceitado o convite de participar da arguição da minha tese e pela disponibilidade, tenho certeza que a presença de vocês trará grandes contribuições a esse trabalho. Aos membros do laboratório do professor Calos Chávez-Olórtegui pela grande ajuda e colaboração durante esse trablho. Aos técnicos de laboratório que passaram pelo LBMM, ICB e servidores que sempre me ajudaram nos experimentos mais complexos, em especial ao Jamil. A toda a família Kalapogrupo (IC’s, técnicos, mestrandos, doutorandos, pós docs etc...), a presença de vocês e companheirismo foi crucial para a boa convivência no laboratório e ajuda nesse trabalho. Em especial gostaria de agradecer ao Lucas, Carolina, Bárbaras que me ajuradam MUITO em todo o desenvolvimento. (desculpe por não citar outros nomes, mas tenho medo de deixar alguém de fora ). Aos meus queridos amigos, que tiveram paciência em aturar minhas ausências e neuroses durante esse período. Pela ajuda nas horas complicadas e pelos momentos de descontração super importantes para que eu não me isolasse do mundo. A toda minha família pelo apoio e compreensão durante esse período. Vocês me ajudaram muito para que eu fosse capaz de escrever e me desenvolver. Em especial aos meus Pais e irmãos que acompanharam mais de perto esse momento. E ao restante da família pelos momentos de descontração em festas e pequenas viagens. VIII Sumário AGRADECIMENTOS ........................................................................................................................................ VII SUMÁRIO ...................................................................................................................................................... VIII ÍNDICE DE ILUSTRAÇÕES ................................................................................................................................. XI ÍNDICE DE TABELAS ........................................................................................................................................ XII LISTA DE ABREVIATURAS .............................................................................................................................. XIII RESUMO .........................................................................................................................................................15 ABSTRACT .......................................................................................................................................................16 1. INTRODUÇÃO .........................................................................................................................................17 1.1 Escorpiões ........................................................................................................................................ 17 1.2 Escorpionismo .................................................................................................................................. 18 1.3 Epidemiologia do Escorpionismo ..................................................................................................... 19 1.3.1 Velho Mundo ................................................................................................................................... 19 1.3.2 Novo Mundo .................................................................................................................................... 20 1.4 Peçonha escorpiônica e toxinas ....................................................................................................... 21 1.5 Soro antipeçonha escorpiônico ........................................................................................................ 23 1.6 Historia da produção de soro antiescorpiônico em cavalos ............................................................ 23 1.7 Formas alternativas de produção de soro antiescorpiônico ............................................................ 24 1.7.1 Imunização com peçonha detoxificada ........................................................................................... 24 1.7.2 Imunização com frações tóxicas da peçonha .................................................................................. 25 1.7.3 Imunização com anatoxinas nativas ................................................................................................ 26 1.7.4 Imunização com toxinas recombinantes ......................................................................................... 26 1.7.5 Imunização com peptídeos sintéticos .............................................................................................. 28 1.7.6 Anticorpos monoclonais contra a peçonha escorpiônica ................................................................ 29 1.7.7 Anticorpos recombinantes baseados em antipeçonha escorpiônica ............................................... 30 1.8 Tityus serrulatus .............................................................................................................................. 31 1.8.1 Epidemiologia de T. serrulatus ........................................................................................................ 33 1.8.2 Toxinas de T. serrulatus ................................................................................................................... 35 1.8.3 Tratamento de escorpionismo ......................................................................................................... 37 1.8.4 Problemas na produção de soro ...................................................................................................... 37 2. JUSTIFICATIVA ........................................................................................................................................40 3. OBJETIVOS ..............................................................................................................................................41 3.1 Objetivos Específicos ........................................................................................................................ 41 4. MATERIAL E MÉTODOS ...........................................................................................................................42 4.1 Animais e linhagens de E.coli utilizadas........................................................................................... 42 4.2 Extração de peçonha de T. serrulatus .............................................................................................. 42 4.3 Busca de clones na Biblioteca de cDNA para a construção de cassetes quiméricos ........................ 43 4.4 Vetores plasmidiais utilizados ......................................................................................................... 43 4.5 Reação em cadeia da Polimerase (PCR)........................................................................................... 43 4.6 Eletroforese em gel de agarose ....................................................................................................... 44 IX 4.7 Transformação em E. coli ................................................................................................................ 44 4.8 Lise alcalina para purificação plasmidial ........................................................................................
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