Caracterização de transcritos de ancilostomídeos envolvidos no parasitismo

• Ano de defesa: 2012
• Autor(a) principal: Ana Flavia Dias Vieira da Costa
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Parasitologia; Ancylostoma; Inibidores de proteases Kunitz; Expressão gênica; Ancilostomídeos; Parasitismo
• Link de acesso: http://hdl.handle.net/1843/BUOS-94MKET

This study evaluated the transcription profile and differentiaL expression of two classes of genes involved in parasitism by hookworms: asps and Kunitz protease inhibitors. These two classes of genes have been previously identified by presenting a higher level of transcription in males of A. braziliense. The differential transcription was assessed by RT-PCR and qRT-PCR, for asps 4, 5 and 6 of A. braziliense and A. caninun, asps 4 and 5 of A. ceylanicum and two Kunitz proteinase inhibitors genesfor the three species. In all species analyzed these genes presented astranscriptionally enriched in male worms. To analyze the different species, new genes were sequenced having been produced seven new partial sequences of asps and Kunitz protease inhibitors genes for the three Ancylostoma species. Since there were no available sequences of these genes for adults N. americanus in the database, 94 consensus sequences were generated "in silico" from the EST database, based on similarity with the orthologous proteins already described for A. ceylanicum and A. caninum. KU domain sequences originating of four different species of hookworms were expressed and used for antibody production. These antibodies were used to compare the differential expression of Kunitz protease inhibitors present in ES products and in total protein extracts between males and females of all four species, by western blot. It was observed a differential expression to some proteins between males and females of A. braziliense A. caninum, A. ceylanicum and N. americanus, and 14 distinct proteins containing the conserveddomain have been recognized, some as specific to ES products of N. americanus. The characterization of these proteins can be addressed in future studies. Furthermore, it was shown that recombinant domains KU did not provide protection in hamsters against infection by A. ceylanicum. However, in view of the potential antigenic profile presented by the domains, it opens the prospect that they can be used in combination with other peptides for vaccination trials. Finally, KU recombinants domains were used to assess the immunogenic potential of proteins produced by hookworms possessing such domains, by ELISA using sera from hamsters and dogs infected or not by hookworms. Only sera from dogs infected with A. caninum was able to recognize the recombinant KU domains.