Clonagem e expressão de uma fosfolipase D recombinante do veneno da aranha Loxosceles gaucho

Abstract

The venom of the Loxosceles sp spiders comprises a mix of diverse toxins which results in an intense local inflammatory reaction, promotes dermonecrotic skin lesion, platelet aggregation, hemolytic anemia, and acute renal failure in rare cases. Among the toxins found in the venom, the phospholipases D (PLDs) also called dermonecrotic toxins, are the most important and well-studied proteins in the venom, since they assemble the main effects observed in loxoscelism. Despite the importance of these toxins, the biological analyses of PLDs are hampered by their small amount obtained in the venom. Therefore, the strategy of cloning and expression of these toxins in recombinant form in the bacterial system has been widely used and it is currently an important approach to obtain a huge amount of these molecules and to study their biological effects. However, despite the importance of Loxosceles gaucho venom, until now, any PLDs from this specie was isolated, cloned or expressed in a recombinant form. Thereby, in this work, we showed the cloning of cDNA that codifies a PLD from L. gaucho venom gland in pAE plasmid. The recombinant protein, named LgRec1 was produced in E. coli and then purified by IMAC, that resulted in a single band with molecular weight of 32kDa and a yield of 3.2 mg/L for culture medium. Analyzes of biological activity of this toxin showed that is able to promote local reaction (edema, erythema, bruising and pallor) and dermonecrosis; induce platelet aggregation; hydrolyze sphingomyelin and cause hemolysis. Neutralization assays using antibodies produced in rabbits to LgRec1 showed to be effective in inhibiting the local reaction (~ 65%) and dermonecrosis (~ 100%) evoked by whole venom, this indicates a potential use of these antibodies in neutralizing the toxic effects of envenomation. Therefore, the data presented in this work could help us understand the mechanism of action of PLDs in the envenomation caused by Loxosceles spider genus.