Heterologous Expression, Purification and Immunoreactivity of the Antigen 5 from Polybia paulista Wasp Venom
Murilo Luiz Bazon,
Amilcar Perez-Riverol,
José Roberto Aparecido dos Santos-Pinto,
Luis Gustavo Romani Fernandes,
Alexis Musacchio Lasa,
Débora Laís Justo-Jacomini,
Mario Sergio Palma,
Ricardo de Lima Zollner,
Márcia Regina Brochetto-Braga
Affiliations
Murilo Luiz Bazon
Laboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP, Univ Estadual Paulista, Av. 24-A, n° 1515, Bela Vista, Rio Claro, SP CEP 13506-900, Brazil
Amilcar Perez-Riverol
Laboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP, Univ Estadual Paulista, Av. 24-A, n° 1515, Bela Vista, Rio Claro, SP CEP 13506-900, Brazil
José Roberto Aparecido dos Santos-Pinto
Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP, Univ Estadual Paulista, Av. 24-A, n° 1515, Bela Vista, Rio Claro, SP CEP 13506-900, Brazil
Luis Gustavo Romani Fernandes
Laboratório de Imunologia Translacional-LIT, Departamento de Clínica Médica, Faculdade de Ciências Médicas, FCM, Universidade Estadual de Campinas-UNICAMP, Rua Tessália Vieira de Camargo n° 126, Cidade Universitária “Zeferino Vaz”, Campinas, SP CEP 13083-887, Brazil
Alexis Musacchio Lasa
System Biology Department, Biomedical Research Division, Center for Genetic Engineering and Biotechnology, Ave. 31, e/ 158 and 190, P.O. Box 6162, Cubanacan, Playa, Havana 10600, Cuba
Débora Laís Justo-Jacomini
Laboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP, Univ Estadual Paulista, Av. 24-A, n° 1515, Bela Vista, Rio Claro, SP CEP 13506-900, Brazil
Mario Sergio Palma
Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP, Univ Estadual Paulista, Av. 24-A, n° 1515, Bela Vista, Rio Claro, SP CEP 13506-900, Brazil
Ricardo de Lima Zollner
Laboratório de Imunologia Translacional-LIT, Departamento de Clínica Médica, Faculdade de Ciências Médicas, FCM, Universidade Estadual de Campinas-UNICAMP, Rua Tessália Vieira de Camargo n° 126, Cidade Universitária “Zeferino Vaz”, Campinas, SP CEP 13083-887, Brazil
Márcia Regina Brochetto-Braga
Laboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP, Univ Estadual Paulista, Av. 24-A, n° 1515, Bela Vista, Rio Claro, SP CEP 13506-900, Brazil
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.
