Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion.
- Author:
Fu-xiang ZHU
1
;
Shu-de YANG
;
Ze-long LIU
;
Jing MIAO
;
Hui-ge QU
;
Xiao-yan CHI
Author Information
1. Life Science College of Ludong University, Yantai 264025, China. fuxiangmail@163.com
- Publication Type:Journal Article
- MeSH:
Animals;
COS Cells;
Cercopithecus aethiops;
Factor VIII;
genetics;
metabolism;
secretion;
Genetic Vectors;
Humans;
Inteins;
Peptide Fragments;
genetics;
metabolism;
secretion;
Plasmids;
Protein Splicing;
Swine;
Trans-Splicing;
Transfection
- From:
Acta Pharmaceutica Sinica
2010;45(10):1232-1238
- CountryChina
- Language:Chinese
-
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.
