2) using the National Center for Biotechnology Information (NCBI) Protein Database. The search parameters were as follows: no restrictions on protein molecular mass, one missed tryptic cleavage allowed, mass tolerance to peptide of 0.2 Da for MS spectra. Carbamide-methylation due to treatment of sulfhydryls with iodoacetamide and oxidation of methionine were specified in MASCOT as fixed and variable modifications, respectively. The Pp-Hyal-specific antibody was prepared in the Experimental Immunology and Allergy Laboratory-LIAE, Medical Clinic Department, UNICAMP, Campinas, SP, Brazil. A total of 12 Balb/c female mice at approximately 30-days-of age and a

weight of 25 g were used in the experiments. From the Pp-Hyal purified sample obtained by ion exchange liquid chromatography, 1 mg of total proteins were separated by 15% SDS-PAGE. As only one 39 kDa band was visualized in check details selleckchem the gel, it was cut out, macerated, diluted in sterile physiological solution and applied to the backs of six mice (approved by the Ethics Committee for Animal Utilization-CEUA-No. 031/2010) to produce the Pp-Hyal-specific antibody. Immunizations were done on day 7, 21, and 28, and on day 30, the animals were sacrificed and the antibody collected. Six mice were used as controls, receiving applications of polyacrylamide

gel free of proteins that had been macerated and diluted as described above. Following SDS-PAGE, venom proteins were transferred to a nitrocellulose

membrane (0.45 μ) at 0.8 mA/cm² and 60 V for 2 h in a semi-dry system (New Blot Multiphor II unit, Biotech Pharmacy). Transfer efficiency was confirmed by staining the gel with Coomassie Blue G-250. Immunodetection was performed with the Pp-Hyal-specific antibody diluted 1:1000 and anti-mouse IgG, alkaline phosphatase conjugate (Sigma–Aldrich, USA) diluted second 1:5000 (2 μL in 10 mL of blocking solution) as the primary and secondary antibodies, respectively. Bands were visualized with alkaline phosphatase/BCIP®/NBT (Sigma–Aldrich, USA). The complete cDNA sequence of Pp-Hyal was determined after sequencing 11 positive clones. A 1315 bp consensus cDNA sequence (GI: 302201582) showed the highest similarity with Hyal from the venoms of the four endemic wasp species of the Northern hemisphere: 90% similarity with P. annularis, 81% with V. vulgaris, Vespula germanica, Vespa magnific, and 80% with Dolichovespula maculata. The primary sequence of the deduced Pp-Hyal mature protein ( Fig. 1) contained 338 amino acid residues (1017 bp) and was rich in the amino acids Asn, Gln, and Lys, with a theoretical pI of 8.77 and a predicted molecular mass of 39,648.8 Da versus the 43,277.0 Da indicated by MS. Fig. 1 shows the location of the forward and reverse primers, the three potentially immunogenic N-glycosylated sites (Asn79, Asn187, and Asn325) and the two disulfide bridges (Cys19–Cys308 and Cys185–Cys197) responsible for stabilization of protein structure.