Navegando por Palavras-chave "Enzimas Digestivas"

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    Construção De Biblioteca De Inibidores Proteicos Em Sistema Phage Display Para Seleção De Inibidores Específicos Para Proteases De Larvas De Mosquito Aedes Aegypti
    (Universidade Federal de São Paulo (UNIFESP), 2017-10-26) Manzato, Veronica De Moraes [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    The mosquito Aedes aegypti is well adapted to urban environment and the major vector of several diseases as dengue, yellow fever, zika and chikungunya flaviviruses. It has been considered a global public health problem because, in the last years, the outbreaks of every 3-5 years of dengue have spread with increased virulence. There is no treatment for these diseases and vaccine has low efficacy, as a result, the prophylaxis remains on vector control but the presence of resistant strains of inseticides and larvicidal in use makes the control a difficult and still unsolved problem. Knowing that the larval stage feeds constantly, we intend to interfere with the larvae development by the inhibition of the digestion proteins. A neutrophil elastase and chymotrypsin inhibitor found in Triatoma infestans eggs named TiPI (Triatoma infestans Pacifastin Inhibitor) had the reactive site (P2-P2’) randomly mutated by the phage display technique which allowed their selection against digestive enzymes of the 4th instar larvae midgut. The selected mutants DNA analyzes evidenced group of 11% possible trypsin inhibitors with Arg or Lys at P1 position, 18.5% possible neutrophil elastase inhibitors with Ala, Leu or Val at P1 and curiously, the major group represented by 47% of the mutants with Gln at P1 and 88.8% with Gln at any mutated position. Fusion phages of ten selected mutants were produced, among them 4 possible trypsin inhibitor, mutants 73 (QRLQ), 154 (LKQL), 161 (KRKQ) and 189 (QRHL), 2 possible chymotrypsin inhibitor 29 (ALAR) and 143 (LLQC), and the 4 most frequently mutants, 4 (SQWH), 5 (QSAM), 6 (HQSL) and 22 (AQVF) and their interactions with proteins of the 4th instar larvae midgut and commercial enzymes were evaluated by Surface Plasmon Resonance. The inconclusive results conduct us to protein expression by Pichia pastoris yeast. The yeast supernatant culture containing recombinant mutants was used in the serine 18 protease inhibition assays. Among the trypsin inhibitors, mutant 73.21 showed high inhibitory activity to trypsin, and the mutant 29.14 inhibits neutrophil elastase. Mutants 4.27, 5.26 and 6.27 presented inhibitory activity for subtilisin A. The results confirm that the TiPI1 phage display library allowed the selection of specific mutants against digestive enzymes of larvae of Ae. aegypti. Our results suggested that these molecules can be use in the development of specific synthetic inhibitors with larvicidal activity for Ae. aegypti.