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

Abstract

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 Ni 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.

Insight into the Functional Diversification of Lipases in the Endoparasitoid Pteromalus puparum (Hymenoptera: Pteromalidae) by Genome-scale Annotation and Expression Analysis

“…c6971 from Ooencyrtus telenomicida has 18 and 16 residues in its β9 loop and lid domain, respectively [12]. For some social wasps and ants, their venom-associated phospholipase A1s (PLA1s) have been observed with both shorten β9 loops and small lid domains, indicating that these lipases lack TAG hydrolytic activity either [52][53][54].…”

Current challenges in molecular diagnostics of insect venom allergy

“…Venomic analysis of P. paulista allowed the identification of three major components that were officially annotated as insect venom allergens (Poly p 1, Poly p 2 and Poly p 5) [56]. Recombinant forms of these allergens were obtained in bacterial and yeast cells and are under evaluation for the rational design of CRD [57,58]. Moreover, PLA1 and antigen 5 from of A. pallens were recently identified and sequenced by our group (unpublished data).…”

Wasp venomic: Unravelling the toxins arsenal of Polybia paulista venom and its potential pharmaceutical applications

“…Despite the great diversity of social Hymenoptera identified in Brazil, no component-defined allergenic materials are available for specific diagnosis of allergy [12,35]. Currently, HVA diagnosis is based on the use of crude venom extracts from native species or commercialized allergens from insects of Northern Hemisphere.…”

“…Proteomic analysis allowed the identification and purification of the three major allergens from P. paulista venom: phospholipase A1 (Poly p 1) [39], hyaluronidase (Poly p 2) [40,41] and antigen 5 (Poly p 5) [42]. These allergens are medium (Poly p 2) to highly abundant (Poly p 1 and Poly p 5) venom proteins [23,35,39,41,43]. The combined use of bioinformatics and proteomic tools as well as molecular biology techniques resulted in the cloning, sequencing and further heterologous expression in E. coli of both Poly p 1 [35] and Poly p 2 [12].…”

“…These allergens are medium (Poly p 2) to highly abundant (Poly p 1 and Poly p 5) venom proteins [23,35,39,41,43]. The combined use of bioinformatics and proteomic tools as well as molecular biology techniques resulted in the cloning, sequencing and further heterologous expression in E. coli of both Poly p 1 [35] and Poly p 2 [12]. Also, nPoly p 5 is currently evaluated for the development of a peptide-based allergy diagnoses system and an engineered vaccine [44].…”