Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion

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-splicng 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·mL⁻¹ and (2.52 ± 0.32) u·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·mL⁻¹, (0.72 ± 0.13) u·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.