XXXV Reunião Anual da SBBqResumoID:8538


Identification, purification and characterization of serine protease inhibitors of Aedes aegypti


Watanabe, R.M.O.1, deMarco, R. 1, Gaio, A.O. 2, Lemos, F.J.A.2 and Tanaka A.S.1



1Departamento de Bioquímica da UNIFESP-EPM, São Paulo, SP. 2Laboratório de Biotecnologia da Universidade Estadual do Norte Fluminense, Campos, RJ. E-mail: renatamidori@grad.unifesp.br


Dengue and dengue hemorrhagic fever are tropical viral diseases which are transmitted by Aedes aegypti mosquito. These diseases occur in more than hundred countries and about 100 million people are infected with dengue virus. For World Health Organization (WHO), the major problem for vectors control is our ignorance about their biology. So, better understanding about the vector reproduction and hematophagy adaptation could help in the development of vaccines or drugs to ectoparasites control. In order to contribute to this area, the present work aims were the purification and the partially characterization of a chymotrypsin and elastase inhibitors from Aedes aegypti. Crude extracts of fat body, hemolymph and entire body of Ae. aegypti were used in the chymotrypsin and neutrophil elastase inhibition assays. The results showed higher inhibitory activities for chymotrypsin and neutrophil elastase in the fat body extract. Mosquito's fat body extracts, after blood feeding (0, 3, 6, 12, 18, 24 and 48 h), were used in the protease inhibition assays. The results showed that the inhibitor activities increased in parallel to the total proteins with a peak at 18 h after blood feeding. The chymotrypsin inhibitor was partially purified using fat body extract of 18 h after feeding by affinity chromatography on chymotrypsin-Sepharose column, ion exchange and reverse phase chromatographies. Purified chymotrypsin inhibitor did not present a N-terminal amino acid sequence by Edman degradation, probable because of low protein amount. The perspectives of this work is to purify the elastase inhibitor and the chymotrypsin inhibitor for primary structure characterization.  Supported by: FAPESP and CNPq.