Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion / 药学学报
Acta Pharmaceutica Sinica
;
(12): 1232-1238, 2010.
Article
in Chinese
| WPRIM
| ID: wpr-354522
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Peptide Fragments
/
Plasmids
/
Swine
/
Factor VIII
/
Transfection
/
Chlorocebus aethiops
/
COS Cells
/
Protein Splicing
/
Bodily Secretions
/
Trans-Splicing
Type of study:
Prognostic study
Limits:
Animals
/
Humans
Language:
Chinese
Journal:
Acta Pharmaceutica Sinica
Year:
2010
Type:
Article
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