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.
Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, CEP 13506-900, Bela Vista, Rio Claro, SP, Brazil. Electronic address: aperezriverol@gmail.com.
Amilcar
A
Perez-Riverol
Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, CEP 13506-900, Bela Vista, Rio Claro, SP, Brazil; Laboratório de Mutagênese Ambiental, Avenida 24-A, nº 1515, Bela Vista, Rio Claro, São Paulo, CEP 13506-900, Brazil. Electronic address: franko_mg@hotmail.com.
Franco Dani
FD
Campos Pereira
Center for Genetic Engineering and Biotechnology, Biomedical Research Division, System Biology Department, Ave. 31, e/ 158 and 190, P.O. Box 6162, Cubanacan, Playa, Havana, 10600, Cuba. Electronic address: alexis.musacchio@cigb.edu.cu.
Alexis
A
Musacchio Lasa
Laboratório de Imunologia Translacional, Faculdade de Ciências Médicas, FCM, Universidade Estadual de Campinas-UNICAMP, Rua Vital Brasil, nº 300, CEP 13083-887, Cidade Universitária "Zeferino Vaz", Campinas, SP, Brazil. Electronic address: luisgrf1982@gmail.com.
Luis Gustavo
LG
Romani Fernandes
Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, CEP 13506-900, Bela Vista, Rio Claro, SP, Brazil. Electronic address: jrbio04@rc.unesp.br.
José Roberto Aparecido Dos
JR
Santos-Pinto
Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, CEP 13506-900, Bela Vista, Rio Claro, SP, Brazil. Electronic address: dmjacomini@gmail.com.
Débora Lais
DL
Justo-Jacomini
Instituto de Pesquisa em Bioenergia (IPBEN) (Univ Estadual Paulista), Av. 24-A, nº1515, CEP 13506-900, Bela Vista, Rio Claro, SP, Brazil. Electronic address: gabriel.azevedo4@outlook.com.
Gabriel
G
Oliveira de Azevedo
Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, CEP 13506-900, Bela Vista, Rio Claro, SP, Brazil. Electronic address: bazonmurilo@gmail.com.
Murilo Luiz
ML
Bazon
Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, CEP 13506-900, Bela Vista, Rio Claro, SP, Brazil. Electronic address: mspalma@rc.unesp.br.
Mario Sergio
MS
Palma
Laboratório de Imunologia Translacional, Faculdade de Ciências Médicas, FCM, Universidade Estadual de Campinas-UNICAMP, Rua Vital Brasil, nº 300, CEP 13083-887, Cidade Universitária "Zeferino Vaz", Campinas, SP, Brazil. Electronic address: zollner@unicamp.br.
Ricardo de Lima
RL
Zollner
Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, CEP 13506-900, Bela Vista, Rio Claro, SP, Brazil; Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP (Univ Estadual Paulista), Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado, Botucatu 18610-307, SP, Brazil. Electronic address: mrbbraga@rc.unesp.br.
Márcia Regina
MR
Brochetto-Braga
D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D054466:Phospholipases A1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D014862:Wasp Venoms / Q000276:immunology*; D014863:Wasps
D000485:Allergens; D018076:DNA, Complementary; D014862:Wasp Venoms; D007073:Immunoglobulin E; D054466:Phospholipases A1
Allergy; Diagnosis; Immunoglobulin E (IgE); Polybia paulista; Recombinant phospholipase A1; Venom