, 1990; Kreil, 1995; Cherr et al., 1996; Stern and Jedrzejas, 2006). The social wasp Polybia paulista (Hymenoptera, Vespidae) is endemic to Southeastern Brazil, especially São Paulo State, and is responsible for many accidents due to their venomous stings. Due to consequent and serious allergic reactions that may develop and lead to anaphylactic shock ( Palma, 2006), the social wasp is thus of great medical importance. Studies of crude extracts of P. paulista

venom by chromatography, SDS-PAGE, and specific assays showed significant levels of hyaluronidase, phospholipase, and proteolytic, selleck hemolytic and myotoxic activities ( Silva et al., 2004). Recently, proteomic analysis by Pinto et al. (2012) detected four different glycoproteic forms of Hyal in P. paulista venom MK0683 concentration and subsequently sequenced and structurally modeled the most abundant form, Hyal III. In order to examine the molecular characteristics and immunogenic potential of the Pp-Hyal venom allergen, the complete cDNA sequence

of another form of this enzyme was obtained, cloned, sequenced and its 3D-protein structural model constructed by comparative modeling. Furthermore, the native form of this Pp-Hyal was purified through high performance chromatography and analyzed by mass spectroscopy. The protein was then used to produce a Pp-specific polyclonal antibody, which was tested by Western blotting to confirm its specificity and immune cross-reactivity with venoms from other Hymenoptera species. P. paulista nests were collected in the city of Rio Claro, SP, Southeastern of Brazil. Insects were anesthetized at low temperature

(−20 °C) and their venom reservoirs were extracted with tweezers. Crude venom extract was prepared from 1000 reservoirs, which were macerated at a 1:1 ratio (reservoir:solvent) with ultra pure water containing 1 mM Cyclic nucleotide phosphodiesterase PMSF (Sigma–Aldrich, USA). The suspension was centrifuged at 10,000 × g for 15 min at 4 °C and Pp-Hyal protein was purified from the freeze-dried supernatant. For immunological assays, venom extracts were prepared by the same method with 100 venom reservoirs from each of the following species of Hymenoptera: P. paulista, Polybia sericea, Polybia ignobilis, Agelaia pallipes pallipes, Polistes lanio lanio, A. mellifera, and Solenopsis invicta. Quantification of total proteins in the extracts and fractions from chromatography was performed by the modified Bradford method using bovine serum albumin (BSA) as a standard (Sedmak and Grossberg, 1977). RNA was extracted from 100 venom reservoirs with TRIzol® reagent (Life Technol, USA) and maintained at −85 °C for 7 days to increase the integrity of the total RNA. cDNA synthesis was performed by RT-PCR of 1 μg of RNA using a kit from Promega® (USA) and an oligo dT primer.