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[Trans-splicing of Cys mutated coagulation factor VIII].
Zhu, Fu-Xiang; Liu, Ze-Long; Miao, Jing; Qu, Hui-Ge; Chi, Xiao-Yan.
Affiliation
  • Zhu FX; Life Science College of Ludong University, Yantai 264025, China. fuxiangmail@163.com
Yao Xue Xue Bao ; 47(6): 734-8, 2012 Jun.
Article de Zh | MEDLINE | ID: mdl-22919720
ABSTRACT
To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.
Sujet(s)
Recherche sur Google
Collection: 01-internacional Base de données: MEDLINE Sujet principal: Fragments peptidiques / Facteur VIII / Épissage des protéines / Cystéine / Mutation Limites: Animals Langue: Zh Journal: Yao Xue Xue Bao Année: 2012 Type de document: Article Pays d'affiliation: Chine
Recherche sur Google
Collection: 01-internacional Base de données: MEDLINE Sujet principal: Fragments peptidiques / Facteur VIII / Épissage des protéines / Cystéine / Mutation Limites: Animals Langue: Zh Journal: Yao Xue Xue Bao Année: 2012 Type de document: Article Pays d'affiliation: Chine