Trans-splicing of Cys mutated coagulation factor VIII / 药学学报
Acta Pharmaceutica Sinica
; (12): 734-738, 2012.
Article
ي Zh
| WPRIM
| ID: wpr-276251
المكتبة المسؤولة:
WPRO
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.
النص الكامل:
1
الفهرس:
WPRIM
الموضوع الرئيسي:
Peptide Fragments
/
Factor VIII
/
Transfection
/
Chlorocebus aethiops
/
Gene Transfer Techniques
/
COS Cells
/
Protein Splicing
/
Cysteine
/
Disulfides
/
Genetic Vectors
المحددات:
Animals
اللغة:
Zh
مجلة:
Acta Pharmaceutica Sinica
السنة:
2012
نوع:
Article