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Previous issue date: 2012-09-04 / O câncer cervical é considerado o segundo tipo de tumor a ocasionar maior número de mortes entre as mulheres no mundo,e ainfecção pelopapilomavirus humano (HPV) éa principalcausarelacionada com o desenvolvimento desta neoplasia. No Brasil, o câncer cervicalproduz uma média de 2,5 óbitos/100.000 indivíduos.Atualmente existem duas vacinas no mercado, baseadas em “Vírus-Like Particles”(VLPs), mas o alto custo inviabiliza seu uso pelos países em desenvolvimento. As VLPs de HPV são produzidasa partir daproteína capsidial L1, contra aqual é conferidaforte imunidade humoral.Uma estratégia vacinalorientada para a prevenção do câncer cervical em humanosé muito importante para a saúde pública, no entanto, como hámuitas pessoas já infectadas peloHPV, uma abordagem terapêutica se torna necessária.Neste trabalho foi proposta a expressão do gene L1 de HPV-16 em células da levedura Pichia pastoris, como base de uma estratégia preventiva e a construção de uma vacina de DNA baseada na proteína E5 do HPV-16, identificadacomo potencialantígeno tumoral para terapia docâncer. O gene L1 otimizado de HPV-16 foi clonado em vetor de expressão pPGK para integração no genoma de P. pastoris, sua transcrição foi confirmada por RT-PCR e a expressão da proteína detectada por dot blot, colony blot e western blot.A vacina de DNA foi construída pela inserçãodo gene E5 de HPV-16 otimizado e adicionado doepítopo AU1, novetor pCIneo.Para estudo inicial da funcionalidadeda construção vacinalpCI-E5sintH16, foi realizadaumaanálise in vitroem célulasHEK-293, através de RT-PCRe western blot.O estudo do gene E5 de HPVcomoantígeno terapêutico,apesar depouco exploradoainda, é bastante relevante. / Cervical cancer is the second type of tumor to cause deaths among women worldwide. The infection of human papillomavirus (HPV) is the leading cause related to the development of this neoplasia.In Brazil, cervical cancer produces an average of 2.5 deaths per 100.000 individuals. There are currently two vaccines on the market, based on Virus-Like Particles, but the high cost (U.S. $ 350.00) prevents its use by developing countries. HPV VLPs are formed from the L1 capsid protein, against which it is given strong humoral immunity. A targeted vaccination strategy for the prevention of cervical cancer in humans is very important to public health;however there are many people already infected with HPV, and therefore a therapeutic approach is needed. In this study was proposed the expression of the L1 gene of HPV-16 in Pichia pastorisyeast cells as the basis for a preventive strategy and the construction of a DNA vaccine based on the HPV-16 E5 protein, identified as a potential tumor antigen for cancer therapy. The optimized L1 gene of HPV-16 was cloned into the expression vector pPGK for integration into the genome of Pichia. The transcript of this gene was confirmed by RT-PCR and L1 protein expression detected by dot blot, colony blot and western blot. The DNA vaccine was constructed by cloning the optimized E5 gene of HPV-16, with the inserted AU1 epitope, in the pCIneo vector. In vitro analysis were performed with HEK-293 cells transfected with the PCI-E5sintH16 vector, including RT-PCR and western blot for the initial evaluation of DNA vaccine functionality. The study of the cancer therapy with the E5 gene of HPV-16 although still unexplored is quite relevant.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufpe.br:123456789/18493 |
| Date | 04 September 2012 |
| Creators | COIMBRA, Eliane Campos |
| Contributors | FREITAS, Antonio Carlos de |
| Publisher | Universidade Federal de Pernambuco, Programa de Pos Graduacao em Inovacao Terapeutica, UFPE, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Breton |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFPE, instname:Universidade Federal de Pernambuco, instacron:UFPE |
| Rights | Attribution-NonCommercial-NoDerivs 3.0 Brazil, http://creativecommons.org/licenses/by-nc-nd/3.0/br/, info:eu-repo/semantics/openAccess |
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