The control of viral infections, mainly those caused by influenza viruses, is of great interest in Public Health. Several studies have shown the presence of active properties in the hemolymph of arthropods, some of which are of interest for the development of new pharmacological drugs. Recently, we have demonstrated the existence of a potent antiviral property in the hemolymph of Lonomia obliqua caterpillars. The aim of this study was to produce an antiviral protein in a baculovirus/Sf9 cell system. The resulting bacmid contains the sequence coding for the antiviral protein previously described by our group. Total RNA from L. obliqua caterpillars was extracted with Trizol and used in the reverse transcription assay with oligo(d)T primer followed by polymerase chain reactions (RT-PCR) with specific primers for the cDNA coding for the antiviral protein, based on the sequence deposited in the GenBank database. Restriction sites were inserted in the cDNA for ligation in the donor plasmid pFastBac1™. The recombinant plasmid was selected in Escherichia coli DH5α and subsequently used in the transformation of E. coli DH10Bac for the construction of the recombinant bacmid. This bacmid was used for the expression of the antiviral protein in the baculovirus/Sf9 cell system. After identifying the protein by western blot, activity tests were performed, showing that the purified recombinant protein was able to significantly reduce viral replication (about 4 logs). Studies on the optimization of the expression system for the production of this antiviral protein in insect cells are in progress.

The apoptosis death is a very important factors in production processes that limited the industrial production of some proteins of economic interest. However, one of the forms to increase the cellular productivity would be inhibit or attenuate the cellular death. Recently we have demonstrated the presence of a potent anti-apoptotic protein in Lonomia obliqua hemolymph which extends the cell culture viability through apoptosis prevention. By the other side, has been reported that mitochondria have one important action in the apoptosis control process, being that mitochondria membrane permeabilization (MMP) can be an important stage in this process. MMP associated or not with the loss of the electrochemical potential of the mitochondria and alteration of the matrix is responsible for the inter membranes protein release (e.g. cytochrome c, the AIF, etc) of cytosol. The result obtained showed that the addition of a protein from Lonomia obliqua hemolymph in the culture lead to a prolongation of the cellular life (3-4 days) and the cells leading a high electrochemical potential of the mitochondria. This protein can has its action in mitochondria membrane, avoiding the loss of the membrane permeability and the Cytochrome-C release. As positive control, apoptosis death in these cultures was induced by 50 μm of t-BHP or 600 μm of H202. The presence of apoptosis was characterized by flow citometry, microscopy electronic and agarose gel electrophoresis. The potential electrochemical of the mitochondria was determined by JC-1, Hoechst 33324 and DIOC6. Cytochrome C was identified in cytosol by an anti-cytochrome antibody.

, meu orientador, pela oportunidade concedida, pela atenção, carinho, conversas, apoio e todos os ensinamentos que levarei para toda minha vida pessoal e profissional. A Prof. Drª Darci Moraes Barros Battesti, pela oportunidade de desenvolver este trabalho no laboratório de Parasitologia do Instituto Butantan e pelo incentivo profissional. Um agradecimento especial a minha grande amiga Kátia, sem a qual eu nunca estaria aqui, pois estava sempre disposta a me ensinar e me ajudou a dar os primeiros passos da minha carreira. Aos amigos de parceria do laboratório: Ana Carolina, Nathália, Kelly e Dalton, pela parceria e por estarem sempre dispostos a me ajudar, conversar, ouvir, aconselhar.

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