Padronização da reação de trans-splicing in vitro em tripanosomas utilizando a técnica de RT-PCR

• Autor(a) principal: Polverari, Fernanda Silva [UNESP]
• Data de Publicação: 2013
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UNESP
• Texto Completo: http://hdl.handle.net/11449/108476
• topic: Biotecnologia; Biologia molecular; Reação em cadeia de polimerase; Polymerase chain reaction

The Trypanosomatidae family includes a large number of protozoan parasites, with importants etiological agents of human diseases. Among the causes of diseases stand out Trypanosoma brucei (responsible for sleeping sickness), Trypanosoma cruzi (Chagas disease causative agent) and Leishmania sp (causing leishmaniasis). These parasites have a mechanism that form their mRNAs, the trans-splicing reaction, which involves the introns (intergenic regions) excision and two exons union of the independent transcripts, RNA SL (spliced leader) and pre-mRNA acceptor. In another laboratory study, the trans-splicing in vitro reaction was established using nuclear extracts (cell-free) from epimastigotes forms of T. cruzi and / or procyclic forms of T. brucei, and as pre-mRNA aceptor was used the alpha tubulin partial sequence (containing the intronic region, splice site and a radiolabelled part of alpha tubulin exon from T. cruzi (unpublished data). The use of radioactive material is a limiting factor, due to its high hazard (extremely harmful to humans and environment), and also by the high cost relative to the current bureaucratic procedures for importation. Therefore, in this work the reaction was reproduced without the use of radioactive material, using the reverse transcription / polymerase chain reaction (RT-PCR) and agarose gel stained with ethidium bromide for analysis of the formed products. The method allows the evaluation of trypanocidal substances interference searching potential therapeutic targets in the parasite ribonucleoprotein, preserving the host cells.