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Francisco Carlos Faria Lobatohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782869H0&tokenCaptchar=03AOLTBLRtMgMiYTKKDEF13BzXOal4fBvd7mDxv6X6453fiGd8JoQgbFSp8l9qCcHnsqjGdt8U47aecKU8zwXzhs6--yh11RdIn5adB2GR0U0PjCSKocF68cRa036GEe4OHcpCQ-SEygt3lmu9xfIxou0ZIootXgSDAR8TcVPxq77wmvlhyjfampg_VoOjqWeFkuZajxCUONmBOuWqiOkDAAJuxvoGKFHB97hgwmKjuINOwWhHQjhPhduaF_4jGAk2o9gO-wgepeMCyeLM2HMJVppTTqcpwZkPHbeoQNRkGTKsmatfl-A_AmGl0ctW1iChNXVJutpwgFmDL7zUIco8EjxjG3bzY25tL1XBS8GRS2zU22TSKc1iW6LwOP0yTFF0EYzs_9c_H4TKZKIcZdOdxrbCxGtcKBmRrgq-PjaG8EANUvc4LQdHt0uSDzlQ2uS3YGnRY-3WB6QCftV7iJUDHpPV9VtgIWCVfXqFY2xvfWWtK9jqanuLsGzd3i-gR2-1tUMeQx1RjNekLuiz Guilherme Dias HeneineRodrigo Otávio Silva SilveiraRicardo Andrez Machado de ÁvilaRonnie Antunes de AssisJenner karlisson Pimenta dos ReisDaniel Menezes SouzaLuiz Guilherme Dias Heneinehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4487684T5&tokenCaptchar=03AOLTBLSVaFPJoNRFRRIMvYGR7xV6KIPZgr6bZMWhGBlveDspiu3KOFNfsRgC7ja4AJyb1hJhyIfyK1mGAxh9Rb3H02lR7WzL0thxNbf62GoTAiaRl9aPqaqwJxUOI_k55ej4lezZR3kFjm_RBZY5efq1EFyHBzT6BdXPeZo6y6-JRM65GRGCsDpZNX84UFjLPP_Wl9JY6qPl7X_jIfXnBpZlVqvj2Qe6MSZv--ejwBejQ2i1pSb7pJYydxuFCos1dJK-qXyPJIPsGA-Gwsei0vKg_n_Mioy7DgZGBkBe7meRFrMBJbIeBM1wPxR-QvjJK0SOU28SL9lYlFMPLA9771CLY9E7TrVQes8fIrhieqKjOGgpEt0n4FcEATqGnMzTJQqc_E6UrbPqAUn6sdWyQoOXjXbniLchKP9u0iMHHrhrK2bgMB5eAzS2e9uhKTEsvhXjW_VOplX1-CxotEOT8slWTcWyvf6kJxtUP_wRmHdnuW_W_pwlmLtYKbePt1pDOl5X2mCikzLZGuilherme Guerra Alves2020-12-31T03:46:37Z2020-12-31T03:46:37Z2017-04-26http://hdl.handle.net/1843/34603O objetivo deste trabalho foi produzir, purificar e avaliar as toxinas e antitoxinas C e D de Clostridium botulinum, beta e épsilon de Clostridium perfringens, bem como compará-las aos padrões existentes utilizados pelos órgãos oficiais, visando suas utilizações em pesquisas para a produção de novas vacinas, desenvolvimento e implementação de métodos alternativos para diagnóstico e controle de vacinas clostridiais. A produção das toxinas foi realizada por meio do cultivo das estirpes em meios específicos para cada espécie em biorreator de bancada. As toxinas C e D de C. botulinum foram purificadas por filtração em gel. A toxina beta de C. perfringens tipo C foi purificada por meio de cromatografia por afinidade metálica, utilizando o zinco como agente quelante. A toxina épsilon de C. perfringens tipo D foi purificada por cromatografia de troca aniônica. As imunoglobulinas G e Y foram produzidas pela múltipla imunização de ovinos e galinhas, respectivamente, com as toxinas purificadas inativadas e conjugadas ao adjuvante de Freund. Posteriormente, as IgG foram purificadas por meio das precipitações por ácido caprílico e sulfato de amônio; e as IgY foram purificadas pela precipitação por sulfato de amônio. As toxinas C e D de C. botulinum foram purificadas ao nível de várias bandas em gel de SDS-PAGE, as quais representam as toxinas, suas subunidades e proteínas auxiliares associadas. A toxina beta de C. perfringens tipo C foi purificada ao nível de uma banda de alta intensidade em gel, e poucas bandas com alto peso molecular e de fraca intensidade que podem se tratar de formas oligomerizadas da toxina. A toxina épsilon de C. perfringens tipo D foi purificada ao nível de uma banda de altíssima intensidade em gel de SDS-PAGE, e poucas bandas de alto peso molecular e de baixíssimas intensidades. As toxinas padrões produzidas por este estudo apresentaram rendimentos proteicos e purezas superiores aos padrões oficiais. As IgG e IgY produzidas e purificadas obtiveram altos títulos, evidenciando a qualidade dos imunógenos e da purificação das imunoglobulinas. Além disso, apresentaram altos índices de avidez, maiores que os índices dos padrões oficiais, evidenciando a qualidade superior dos insumos produzidos por este estudo. O presente trabalho produziu insumos, toxinas e antitoxinas clostridiais, com altos graus de pureza e de qualidade superior aos insumos oficiais nacionais, podendo ser utilizados para o desenvolvimento de novos métodos diagnósticos, vacinas e metodologias de controle da qualidade das vacinas.The objective of this work was to produce, purify and analyze the toxins and antitoxins C and D from Clostridium botulinum, beta and epsilon from Clostridium perfringens, as well as to compare them to the existing standards used by the official organs, aiming their uses in research for the production of new vaccines, development of alternative diagnostic methods and quality control of vaccines. The production of the toxins was carried out by fermentation of the strains in specific media for each species in a bench bioreactor. C. botulinum C and D toxins were purified by gel filtration. C. perfringens type C beta toxin was purified by immobilized-metal affinity chromatography using zinc as a chelating agent. C. perfringens type D epsilon toxin was purified by anion exchange chromatography. Immunoglobulins G and Y were produced by multiple immunization of sheep and chickens, respectively, with inactivated purified toxins in Freund's adjuvant. Subsequently, IgG were purified by precipitation by caprylic acid and ammonium sulfate, and the IgY were purified by ammonium sulfate precipitation. C. botulinum C and D toxins were purified to the level of several SDS-PAGE gel bands, which represent the toxins, their subunits and accessory proteins associated. C. perfringens type C beta toxin was purified to the level of a high intensity gel band, and few bands of high molecular weight and low intensity which may be oligomerized forms of the toxin. C. perfringens type D epsilon toxin was purified to the level of a very high intensity gel band, and few bands of high molecular weight and very low intensities. The standard toxins produced by this study showed higher yields of protein and higher purity when compared to the official standards. The produced and purified IgG and IgY obtained high titers, revealing the quality of the immunogens and purification methods. In addition, they presented high avidity indexes, higher than the indexes of the official standards, indicating the superior quality of the products generated by this study. The present work produced clostridial toxins and antitoxins with high purity levels and superior quality when compared to the official national ones, and they can be applied for the development of new diagnostic methods, vaccines and methodologies for quality control of vaccines.CNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas GeraisCAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorporUniversidade Federal de Minas GeraisPrograma de Pós-Graduação em Ciência AnimalUFMGBrasilVET - DEPARTAMENTO DE MEDICINA VETERINÁRIA PREVENTIVAhttp://creativecommons.org/licenses/by-nc-nd/3.0/pt/info:eu-repo/semantics/openAccessClostridiumAnticorpoImunoglobulinaGalinhaToxinasProdução e purificação de toxinas e antitoxinas clostridiaisinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMGORIGINALTese Doutorado vfinal.pdfTese Doutorado vfinal.pdfapplication/pdf2935016https://repositorio.ufmg.br/bitstream/1843/34603/1/Tese%20Doutorado%20vfinal.pdf9f0dc3cec686a66f633c7cd52ebf48e0MD51CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufmg.br/bitstream/1843/34603/2/license_rdfcfd6801dba008cb6adbd9838b81582abMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82119https://repositorio.ufmg.br/bitstream/1843/34603/3/license.txt34badce4be7e31e3adb4575ae96af679MD531843/346032020-12-31 00:46:37.735oai:repositorio.ufmg.br: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Repositório InstitucionalPUBhttps://repositorio.ufmg.br/oaiopendoar:2020-12-31T03:46:37Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
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