title: [Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion].
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.
authors.affiliation: Life Science College of Ludong University, Yantai 264025, China. fuxiangmail@163.com
authors.forename: Fu-xiang
authors.initials: FX
authors.lastname: Zhu
authors.forename: Shu-de
authors.initials: SD
authors.lastname: Yang
authors.forename: Ze-long
authors.initials: ZL
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Miao
authors.forename: Hui-ge
authors.initials: HG
authors.lastname: Qu
authors.forename: Xiao-yan
authors.initials: XY
authors.lastname: Chi
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D047668:Inteins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D019154:Protein Splicing; D013552:Swine; D020040:Trans-Splicing; D014162:Transfection
chemical_list: C451034:B-domain-deleted factor VIII; D010446:Peptide Fragments; D005169:Factor VIII