维普中文期刊产品整合服务

The effect of a secretion-enhanced heavy chain on improving intein-based dual-vector co-delivery of a full-length factor Ⅷ gene

查看全文 作　　者：ZHU FuXiang YANG ShuDe LIU ZeLong MIAO Jing QU HuiGe CHI [1]XiaoYan 高影响力作者 机构地区：[1]College of Life Sciences, Ludong University, Yantai 264025, China高影响力机构 出　　处：《Chinese Science Bulletin》索引2011年第56卷第2期,共6页高影响力期刊 基　　金：supported by the Natural Science Foundation of Shandong Province,China(ZR2010CM061);the Science and Technology Program of Yantai(2008152);the Scientific Research Foundation of Ludong University(LZ20083305) 摘　　要：Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene,which is difficult to package in the promising adeno-associated virus (AAV) vectors.Inhibited secretion of FVIII is caused mainly by inefficient secretion of its heavy chain.Previously,we have employed a protein splicing-based dual-vector to co-transfer a B-domain-deleted FVIII (BDD-FVIII) gene,suggesting that the light chain,covalently ligated to a co-expressed heavy chain can improve the secretion of spliced BDD-FVIII.However,its level of secretion was affected by inefficient secretion the heavy chain.Here,we studied the effect of a mutant heavy chain with L303E/F309S substitutions,which enhance FVIII secretion on the heavy chain itself and spliced FVIII when using a protein splicing-based split-delivery of a full-length FVIII gene.Eukaryotic vectors expressing Ssp DnaB intein-fused mutant heavy and light chains were transiently co-transfected into cultured COS-7 cells.A spliced FVIII protein was seen in co-transfected cells by Western blot analysis.The heavy chain was secreted by cells transfected with the mutant heavy chain gene alone at (39±11) ng/mL and this secretion increased to (123±13) ng/mL when cells were co-transfected with the light chain gene,which was greater than the secretion of wild-type heavy chain.The amount of spliced FVIII in the culture supernatant of co-transfected cells was (86±14) ng/mL,with an activity of (0.61±0.08) IU/mL,which was greater than that of wild-type FVIII co-transfected cells.Spliced FVIII and bioactivity were also detected in the combined culture supernatant of cells individually transfected with mutant heavy and light chain gene at a higher level than that of combined wild-type heavy and light chain transfections.This suggested that the heavy chain with improved secretion markedly increased the efficacy of protein splicing-based split delivery of the full-length FVIII gene using a dual-vector.These results encourage the transfer of this technology to an animal model using a dual-AAV vector. 关 键 词：分泌作用 基因传递 因子和 内含肽 轻链 载体 基础 蛋白质剪接
相关文献

参考文献(20)

引证文献(8)

耦合文献(26)

网站首页 | 关于我们 | 联系我们 | 产品服务 | 客服中心 | 广告服务 | 版权声明 | 网站联盟 | 友情链接 | 售卡网点

版权所有© 渝B2-20050021-1　渝公网安备 50019002500403号　违法和不良信息举报中心

互联网出版许可证 新出网证(渝)字10号 全国400电话 - 免长途话费