Protein trans-splicing based dual-vector delivery of the coagulation factor VIII gene.
A dual-vector system was explored for the delivery of the coagulation factor VIII gene, using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally. A pair of eukaryotic expression vectors, expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII), was constructed. With transient co-transfection of the two vectors into 293 and COS-7 cells, the culture supernatants contained (137+/-23) and (109+/-22) ng mL(-1) spliced BDD-FVIII antigen with an activity of (1.05+/-0.16) and (0.79+/-0.23) IU mL(-1) for 293 and COS-7 cells, respectively. The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes. The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting. The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.
Zhu
FuXiang
F
Life Science College of Ludong University, Yantai, 264025, China. fuxiangmail@163.com
Liu
ZeLong
Z
Chi
XiaoYan
X
Qu
HuiGe
H
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D014162:Transfection
C451034:B-domain-deleted factor VIII; D017931:DNA Primers; D010446:Peptide Fragments; D005169:Factor VIII