Construction of expression vectors for eosinophil granule proteins expression and their application in the study of platelet inhibitory effect (2005)
- Authors:
- Autor USP: NUCCI, GILBERTO DE - ICB
- Unidade: ICB
- Assunto: FARMACOLOGIA
- Language: Português
- Abstract: Objetivo: Some authors have observed that eosinophilia can be associated with reduction of platelet aggregation. Our previous results showed that rat eosinophil extract dose dependently inhibited platelet aggregation. Mass spectrometric analysis using Q-TOF showed the presence of proteins with a molecular weight ranging from 12 to 20 kDa. Many of these polypeptide chains were identified as the specific eosinophil granule proteins, MBP, EPO, EDN and ECP. Our objectives were to clone the cDNA for all four proteins to express them in both prokaryotic and eukaryotic cells. Métodos e Resultados: cDNAs were obtained by RT-PCR. Messenger RNAs were obtained from HL60 clone 15 cells induced by 0.5 mM butyric acid during 7 days. Primers for the PCR reaction were constructed containing BamHI and XbaI restriction sites at the 5` and 3` edges, respectively. cDNA for MBP contained 670bp and was inserted in the pCDNA3.1+ expression vector. Protein expression has been performed after transient transfection using the U937 cells cultured in RPMI-1640. Due to high homology, EDN and ECP were cloned using the same primers. cDNA for both EDN and ECP contained 487bp and were inserted in the same expression vector. EPO is a longer protein and its cDNA will be obtained using long PCR reactions. For insertion in the prokaryotic expression vector pET28a, primers for all cDNAs were constructed containing BamHI and EcoRI restriction sites at the 5` and 3` edges respectively. Prokaryotic expressionhas been performed using TOP10 E. coli strain. Conclusões: This fast and simple method of cDNA cloning showed to be efficient for the short eosinophil proteins. Our next step is to identify the active protein and to obtain the truncated or point mutated polypeptide chains to characterize the platelet inhibitory domain
- Imprenta:
- Publisher: Federação de Sociedades de Biologia Experimental
- Publisher place: Águas de Lindóia, São Paulo
- Date published: 2005
- Source:
- Título do periódico: Resumos
- Conference titles: Reunião Anual da Federação de Sociedades de Biologia Experimental, FeSBE
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ABNT
MAZIERO, A. M. e DONATO, J. L. e DE NUCCI, Gilberto. Construction of expression vectors for eosinophil granule proteins expression and their application in the study of platelet inhibitory effect. 2005, Anais.. Águas de Lindóia, São Paulo: Federação de Sociedades de Biologia Experimental, 2005. . Acesso em: 19 abr. 2024. -
APA
Maziero, A. M., Donato, J. L., & De Nucci, G. (2005). Construction of expression vectors for eosinophil granule proteins expression and their application in the study of platelet inhibitory effect. In Resumos. Águas de Lindóia, São Paulo: Federação de Sociedades de Biologia Experimental. -
NLM
Maziero AM, Donato JL, De Nucci G. Construction of expression vectors for eosinophil granule proteins expression and their application in the study of platelet inhibitory effect. Resumos. 2005 ;[citado 2024 abr. 19 ] -
Vancouver
Maziero AM, Donato JL, De Nucci G. Construction of expression vectors for eosinophil granule proteins expression and their application in the study of platelet inhibitory effect. Resumos. 2005 ;[citado 2024 abr. 19 ] - Presence of nitric oxide synthase inhibitor activity in rat pleural exsudates
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