Molecular cloning, expression and IgE-immunoreactivity of phospholipase A1, a major allergen from Polybia paulista (Hymenoptera: Vespidae) venom.

Polybia paulista (Hymenoptera: Vespidae) is a clinically relevant social wasp that frequently causes stinging accidents in southeast Brazil. To date, diagnosis and specific immunotherapy (SIT) of allergy are based on the use of crude venom extracts. Production of recombinant forms of major allergens from P. paulista venom will improve diagnosis and SIT of allergic patients by reducing the incidence of cross-reactivity and non-specific sensitization. Here, we describe the molecular cloning, heterologous expression, purification and IgE-mediated immunodetection of phospholipase A1 (Poly p 1), a major allergen from P. paulista venom. The cDNA of Poly p 1 was extracted from venom glands and then cloned, and further expression of the recombinant allergen (rPoly p 1) was achieved in Escherichia coli BL21 (DE3) cells. Purification of rPoly p 1 was performed using immobilized Ni2+ metal affinity chromatography. Also, a single-step chromatographic method allowed the purification of native Poly p 1 (nPoly p 1) from the wasp's venom glands. We used western blotting to evaluate IgE-reactivity of the sera from 10 P. paulista venom-allergic patients to rPoly p 1 and nPoly p 1. High levels of insoluble rPoly p 1 were obtained during heterologous expression. After solubilization of inclusion bodies and purification of the recombinant protein, a unique band of ∼34 kDa was detected in SDS-PAGE analysis. Allergen-specific IgE (sIgE) from allergic patients' sera recognized rPoly p 1, nPoly p 1 and crude venom extract to a similar extent. Our results showed that rPoly p 1 could be used for development of component-resolved diagnosis (CRD) and molecular-defined SIT of P. paulista venom allergy.

Reactivity of IgE to the allergen hyaluronidase from Polybia paulista (Hymenoptera, Vespidae) venom.

To date, there are no allergenic extracts or components available in Brazil to diagnosis and treatment of patients with venom allergy from social wasp (Vespidae Family; Polistinae Subfamily) despite of the great number of existing species. We evaluated the immunogenic potential of the Hyal recombinant protein (Pp-Hyal-rec) which was expressed in an insoluble form in comparison with the allergenic native protein (Pp-Hyal-nat) for recognition of immunoglobulin E (IgE) in the serum of allergic patients to venom of the endemic social wasp Polybia paulista from São Paulo State, Brazil. Hyal cDNA from the venom of the social wasp P. paulista (Pp-Hyal) (GI: 302201582) was cloned into the expression vector pET-28a in Escherichia coli DE3 (BL21) cells. Solubilization and purification of Pp-Hyal-rec from inclusion bodies were performed using Ni(2+) affinity chromatography (Ni-NTA-Agarose) under denaturing conditions. Both the native (Pp-Hyal-nat) and the recombinant (Pp-Hyal-rec) purified allergens were used for Western blotting to assess the levels of Pp-Hyal-IgE specific in the serum of 10 patients exclusively reactive to the venom of the social wasp P. paulista. The immune sera specifically recognized the band corresponding to the Pp-Hyal-rec protein (40 kDa) at a higher intensity than the native allergen (39 kDa). The sera recognized other proteins in P. paulista crude venom extract to a lesser extent, likely corresponding to other venom allergens such as phospholipase (34 kDa), Antigen 5 (25 kDa), and proteases. The recognition pattern of the immune sera to the Pp-Hyal-rec allergen strongly suggests that this recombinant antigen could be used for developing a diagnostic allergy test as well as for specific immunotherapy (IT).

Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis.

In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/ß)(7) barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients.