Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion / 药学学报
Acta Pharmaceutica Sinica
; (12): 1232-1238, 2010.
Article
em Zh
| WPRIM
| ID: wpr-354522
Biblioteca responsável:
WPRO
ABSTRACT
This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.
Texto completo:
1
Índice:
WPRIM
Assunto principal:
Fragmentos de Peptídeos
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Plasmídeos
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Suínos
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Fator VIII
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Transfecção
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Chlorocebus aethiops
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Células COS
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Processamento de Proteína
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Secreções Corporais
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Trans-Splicing
Tipo de estudo:
Prognostic_studies
Limite:
Animals
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Humans
Idioma:
Zh
Revista:
Acta Pharmaceutica Sinica
Ano de publicação:
2010
Tipo de documento:
Article