Trans-splicing of Cys mutated coagulation factor VIII / 药学学报
Acta Pharmaceutica Sinica
;
(12): 734-738, 2012.
Artículo
en Chino
| WPRIM
| ID: wpr-276251
ABSTRACT
To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Fragmentos de Péptidos
/
Factor VIII
/
Transfección
/
Chlorocebus aethiops
/
Técnicas de Transferencia de Gen
/
Células COS
/
Empalme de Proteína
/
Cisteína
/
Disulfuros
/
Vectores Genéticos
Límite:
Animales
Idioma:
Chino
Revista:
Acta Pharmaceutica Sinica
Año:
2012
Tipo del documento:
Artículo
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