vWF improves secretion and activity of intein spliced BDD-FVIII.
- 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:
Factor VIII;
genetics;
metabolism;
secretion;
Genetic Therapy;
Genetic Vectors;
HEK293 Cells;
Hemophilia A;
therapy;
Humans;
Inteins;
Peptide Fragments;
genetics;
metabolism;
secretion;
Plasmids;
Protein Splicing;
Trans-Splicing;
Transfection;
von Willebrand Factor;
genetics;
metabolism;
physiology
- From:
Acta Pharmaceutica Sinica
2010;45(5):595-600
- CountryChina
- Language:Chinese
-
Abstract:
As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.
