Abstract
Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.
Highlights
Brazil hosts a wide diversity of clinically important Neotropical Hymenoptera including wasps, honeybee and ants
The results described here strongly suggest that rPoly p 5 is a potential candidate for development of molecular diagnosis of P. paulista venom allergy
Despite the wide diversity of Hymenoptera identified in regions, the diagnosis is based on skinistests and vitro sIgEand detection extracts, Neotropical regions, of theallergy diagnosis of allergy based oninskin tests in vitrousing specific IgE (sIgE) venom detection using which is related to significant levels of cross-reactivity
Summary
Brazil hosts a wide diversity of clinically important Neotropical Hymenoptera including wasps, honeybee and ants. In the case of wasps, more than 320 species have been currently identified and several have been informed to coexist with human populations [1]. The venoms of these insects are mix of toxins including allergens that could induce local and systemic allergic reactions including life threatening anaphylaxis [2]. In Brazil, the diagnosis of Hymenoptera venom allergy (HVA) has been based in the use of crude venom extracts and is often hampered by the incidence of cross-reactivity caused by the presence of cross-reactive carbohydrate determinants (CCDs) in allergen structures [4]. Molecular diagnosis based on the use of purified and extensively characterized recombinant allergens could help to overcome this pitfall resulting in proper identification of the primary sensitizing venom [5]
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