Cys突变体凝血Ⅷ因子的蛋白质反式剪接  

Trans-splicing of Cys mutated coagulation factor Ⅷ

作　　者：朱甫祥[1] 刘泽隆[1] 缪静[1] 屈慧鸽[1] 迟晓艳[1] 

机构地区：[1]鲁东大学生命科学学院,山东烟台264025

出　　处：《药学学报》2012年第6期734-738,共5页Acta Pharmaceutica Sinica

基　　金：山东省自然科学基金资助项目(ZR2010CM061);教育部留学回国人员科研启动基金项目(20071108)

摘　　要：为改善蛋白质反式剪接效率,将B-区缺失型FⅧ(BDD-FⅧ)重链的Tyr664和轻链的Thr1826突变为Cys,双载体共转COS-7细胞,观察了细胞内蛋白质剪接、二硫键形成和细胞培养上清液中分泌的剪接BDD-FVIII量和活性。Western blotting检测结果显示,Cys突变可在细胞内形成链间二硫键,剪接BDD-FVIII蛋白量明显增加;双夹心ELISA检测分泌的剪接BDD-FVIII为(128±24)ng.mL/1,明显高于对照(89±15)ng.mL/1;Coatest法检测结果显示,细胞分泌的凝血活性为(0.94±0.08)u.mL/1,也明显高于对照(0.62±0.15)u.mL/1。结果表明,链间二硫键形成可显著提高基于蛋白质剪接的双载体转BDD-Ⅷ基因作用,为进一步动物体内实验提供了依据。To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing,we introduce a Cys point mutation at Tyr664 in heavy chain and at Thr1826 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.

关 键 词：凝血Ⅷ因子 蛋白质反式剪接 双载体 转基因 Cys突变 

分 类 号：R963[医药卫生—微生物与生化药学]