Expressão heteróloga, purificação e análise da imunoreatividade do alérgeno antígeno 5 do veneno de Polybia paulista (Hymenoptera, Vespidae)
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Data
2017-02-23
Autores
Bazon, Murilo Luiz [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A vespa social Polybia paulista é muito agressiva e abundante nos Estados de São Paulo e Sul de Minas Gerais e, responsável por muitos acidentes de importância médica. Os principais alérgenos (Fosfolipase, Hialuronidase e Antígeno 5) de seu veneno já foram caracterizados por estudos proteômicos porém, os estudos em nível molecular estão em andamento pelo nosso grupo. O Antígeno 5 (Ag 5) (~23 kDa) tem sido relatado, em alguns venenos de vespas, como um dos mais abundantes e importantes alérgenos, com elevada prevalência de ligação a IgE (em Vespula vulgaris e em Polistes versicolor) enquanto que em outros, como uma molécula hipoalergênica (em Polybia scutellaris). No Brasil, ainda não existem extratos alergênicos ou componentes padronizados para o tratamento e diagnóstico de alergia ao veneno de Hymenoptera. Neste trabalho, o Ag 5 de P. paulista foi clonado, sequenciado, expresso utilizando a levedura Pichia pastoris X-33, purificado e analisado mediante a imunodetecção por IgE. Os maiores níveis de expressão da proteína recombinante rPoly p 5 foram obtidos em meio BMMY, a 28ºC após 120 horas de indução com metanol. O alérgeno recombinante (rPoly p 5), obtido no meio extracelular, foi purificado por cromatografia de afinidade em resina de Ni+2 e posteriormente por exclusão molecular em coluna de Sephadex-G 100 acoplada a um sistema AKTA-FPLC, com um rendimento de 158 μg/mL. Em paralelo, o Ag 5 nativo (nPoly p 5) foi extraído de glândulas de veneno de P. paulista e purificado através de cromatografia de troca catiônica. Análise de Western Blotting foi realizada para avaliar a reatividade IgE dos soros de 10 pacientes alérgicos ao veneno de P. paulista para rPoly p 5 e nPoly p 5. Os resultados obtidos mostraram que P. pastoris é um sistema muito viável para a produção heteróloga do alérgeno rPoly p 5 e que ambas as formas desse alérgeno foram capazes de se ligar à IgE de todos os soros de pacientes alérgicos testados. Portanto, os dados aqui encontrados podem ser utilizados para melhoria no diagnóstico e imunoterapia específica de alergia ao veneno de Polybia paulista.
The social wasp Polybia paulista is very aggressive and abundant in the states of São Paulo and South of Minas Gerais, responsible for many accidents of medical importance. The major allergens (Phospholipase, Hyaluronidase and Antigen 5) of its venom have already been characterized by proteomic studies. However, studies at the molecular level are in progress by our group. Antigen 5 (Ag 5) (~ 23 kDa) has been reported in some wasp venoms as one of the most abundant and important allergens with a high prevalence of IgE binding (in Vespula vulgaris and in Polistes versicolor), while in others as a hypoallergenic molecule (in Polybia scutellaris). In Brazil, there are no standardized allergenic extracts or components for treatment and diagnosis of allergy to Hymenoptera venoms. In this work, Ag 5 from P. paulista was cloned, sequenced, expressed using the Pichia pastoris X-33 yeast, purified and analyzed by immunodetection by IgE. The highest levels of expression of the rPoly p 5 recombinant protein were obtained in BMMY medium at 28 ° C after 120 hours of methanol induction. The recombinant allergen (rPoly p 5) obtained in the extracellular medium was purified by Ni+2 resin affinity chromatography and subsequently by column exclusion of Sephadex-G 100 coupled to an AKTA-FPLC system with an yield of 158 μg/mL. In parallel, the native Ag 5 (nPoly p 5) from P. paulista venom glands was purified by cation exchange chromatography. Western Blotting analysis was used to evaluate the IgE reactivity of the sera from 10 patients allergic to the P. paulista venom for rPoly p 5 and nPoly p 5. The results obtained showed that P. pastoris is a very feasible system for the heterologous production of rPoly p 5 and that both forms of this allergen were able to bind to IgE from all sera of allergic patients tested. We conclude that data found here can be used to improve the diagnosis and specific immunotherapy of allergy to Polybia paulista venom.
The social wasp Polybia paulista is very aggressive and abundant in the states of São Paulo and South of Minas Gerais, responsible for many accidents of medical importance. The major allergens (Phospholipase, Hyaluronidase and Antigen 5) of its venom have already been characterized by proteomic studies. However, studies at the molecular level are in progress by our group. Antigen 5 (Ag 5) (~ 23 kDa) has been reported in some wasp venoms as one of the most abundant and important allergens with a high prevalence of IgE binding (in Vespula vulgaris and in Polistes versicolor), while in others as a hypoallergenic molecule (in Polybia scutellaris). In Brazil, there are no standardized allergenic extracts or components for treatment and diagnosis of allergy to Hymenoptera venoms. In this work, Ag 5 from P. paulista was cloned, sequenced, expressed using the Pichia pastoris X-33 yeast, purified and analyzed by immunodetection by IgE. The highest levels of expression of the rPoly p 5 recombinant protein were obtained in BMMY medium at 28 ° C after 120 hours of methanol induction. The recombinant allergen (rPoly p 5) obtained in the extracellular medium was purified by Ni+2 resin affinity chromatography and subsequently by column exclusion of Sephadex-G 100 coupled to an AKTA-FPLC system with an yield of 158 μg/mL. In parallel, the native Ag 5 (nPoly p 5) from P. paulista venom glands was purified by cation exchange chromatography. Western Blotting analysis was used to evaluate the IgE reactivity of the sera from 10 patients allergic to the P. paulista venom for rPoly p 5 and nPoly p 5. The results obtained showed that P. pastoris is a very feasible system for the heterologous production of rPoly p 5 and that both forms of this allergen were able to bind to IgE from all sera of allergic patients tested. We conclude that data found here can be used to improve the diagnosis and specific immunotherapy of allergy to Polybia paulista venom.
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Palavras-chave
Alérgeno recombinante, Polybia paulista, Expressão heteróloga, Vespa, Pichia pastoris