Author(s): Zhu F, Liu Z, Chi X, Qu H
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Abstract 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.
This article was published in Sci China Life Sci
and referenced in Biochemistry & Physiology: Open Access