Análise imunológica comparada da eficiência e especificidade da hialuronidase recombinante de Polybia paulista (Hymenoptera, Vespidae) expressa em bactéria e levedura
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2013-05-03
Autores
Jacomini, Débora Laís Justo [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A vespa social Polybia paulista tem sido muito estudada nos campos da bioquímica, proteômica e imunologia pelas suas características de habitat, composição de seu veneno e pelas conseqüencias dos acidentes decorrentes de suas ferroadas. O cDNA de hialuronidase (GI: 302201582), um dos principais alérgenos desse veneno, foi clonado em vetores de expressão - pET-28a e pPICZA - em bactéria Escherichia coli DE3 (BL21) e em levedura Pichia pastoris, respectivamente. A proteína Hyal recombinante (Pp-Hyal-rec) de bactéria foi expressa em corpúsculos de inclusão, enquanto que a de levedura na forma solúvel. Ambas foram purificadas por cromatografia de afinidade em resina Ni2+ (Ni-NTA-Agarose). A proteína nativa de Hyal (Pp-Hyal-nat) também foi obtida e purificada até a homogeneidade por meio de cromatografia de troca catiônica em coluna Hiprep FF CM, acoplado a um sistema de Akta FPLC, e as análises realizadas por espectrometria de massa em MALDI ToF/ToF-MS. Anticorpos policlonais foram produzidos em camundongos BALB/c contra a Pp-Hyal-nat e a Pp-Hyal-rec de bactéria, demonstrando alta especificidade nos testes de imunoblotting realizados. Estes alérgenos foram avaliados quanto ao reconhecimento de imunoglobulina E (IgE) Pp-Hyal-específico no soro de pacientes sabidamente alérgicos ao veneno desta vespa. Os soros imunes foram capazes de reconhecer especificamente, em maior intensidade as bandas correspondentes à proteína Pp-Hyal-rec de bactéria (43 kDa) e a Pp-Hyal-rec de levedura (37 kDa) em relação ao alérgeno Pp-Hyal-nat (39 kDa). No extrato de veneno bruto de P. paulista, os soros reconheceram outras proteínas provavelmente correspondentes aos demais alérgenos do veneno, tais como Fosfolipase (37 kDa), Antígeno 5 (25 kDa), e proteases. Os dados aqui obtidos com ambos...
The social wasp Polybia paulista has been well studied in fields of biochemistry, proteomics and immunology due to its habitat characteristics, venom composition and the consequences of accidents arising from their stings. The hyaluronidase cDNA (GI: 302201582), one of the major venom allergens, was cloned into expression vectors - pET-28a and pPICZA - in Escherichia coli DE3 (BL21) and yeast Pichia pastoris, respectively. The recombinant protein Hyal (Pp-Hyal-rec) of bacteria was expressed in inclusion corpuscles, whereas the yeast in soluble form. Both were purified by affinity chromatography on Ni2+ (Ni-NTA-Agarose) resin. The native protein Hyal (Pp-Hyal-nat) was also obtained and purified to homogeneity by cation exchange chromatography on Hiprep CM FF column, to an Akta FPLC coupled system, and the analysis performed by mass spectrometry MALDI ToF / ToF-MS. Polyclonal antibodies were produced in BALB/c mice against Pp-Hyal-nat and Pp-Hyal-rec from bacteria, demonstrating an high specificity in the immunoblotting tests performed. These allergens were evaluated for recognition of immunoglobulin E (IgE) Pp-Hyal-specific in serum from patients known to be allergic to the venom of this wasp. The immune sera were able to recognize specifically in a higher intensity the bands corresponding to protein Pp-Hyal-rec of bacteria (43 kDa) and Pp-Hyal-rec of yeast (37 kDa) in relation to the allergen Pp-Hyal-nat (39 kDa). In the P. paulista crude venom extract, all sera recognized other proteins probably corresponding to other venom allergens, such as phospholipase (37 kD), antigen 5 (25 kDa), and protease. The data obtained here with both recombinant allergens strongly suggest the possibility of using... (Complete abstract click electronic access below)
The social wasp Polybia paulista has been well studied in fields of biochemistry, proteomics and immunology due to its habitat characteristics, venom composition and the consequences of accidents arising from their stings. The hyaluronidase cDNA (GI: 302201582), one of the major venom allergens, was cloned into expression vectors - pET-28a and pPICZA - in Escherichia coli DE3 (BL21) and yeast Pichia pastoris, respectively. The recombinant protein Hyal (Pp-Hyal-rec) of bacteria was expressed in inclusion corpuscles, whereas the yeast in soluble form. Both were purified by affinity chromatography on Ni2+ (Ni-NTA-Agarose) resin. The native protein Hyal (Pp-Hyal-nat) was also obtained and purified to homogeneity by cation exchange chromatography on Hiprep CM FF column, to an Akta FPLC coupled system, and the analysis performed by mass spectrometry MALDI ToF / ToF-MS. Polyclonal antibodies were produced in BALB/c mice against Pp-Hyal-nat and Pp-Hyal-rec from bacteria, demonstrating an high specificity in the immunoblotting tests performed. These allergens were evaluated for recognition of immunoglobulin E (IgE) Pp-Hyal-specific in serum from patients known to be allergic to the venom of this wasp. The immune sera were able to recognize specifically in a higher intensity the bands corresponding to protein Pp-Hyal-rec of bacteria (43 kDa) and Pp-Hyal-rec of yeast (37 kDa) in relation to the allergen Pp-Hyal-nat (39 kDa). In the P. paulista crude venom extract, all sera recognized other proteins probably corresponding to other venom allergens, such as phospholipase (37 kD), antigen 5 (25 kDa), and protease. The data obtained here with both recombinant allergens strongly suggest the possibility of using... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Wasps, Enzimas, Vespa, Alergenos, Veneno, Imunologia, Imunologia comparada
Como citar
JACOMINI, Débora Laís Justo. Análise imunológica comparada da eficiência e especificidade da hialuronidase recombinante de Polybia paulista (Hymenoptera, Vespidae) expressa em bactéria e levedura. 2013. 85 f. Tese - (doutorado) - Universidade Estadual Paulista, Instituto de Biociências de Rio Claro, 2013.