A1区替换提高人BDD-FⅧ重链的分泌并缓解其与轻链共转基因细胞链分泌的不均衡性  

Substitution of A1 Domain Enhances Secretion of Human BDD-FⅧ Heavy Chain and Relieves Chain Imbalance Secreted by the Heavy and Light Chain Co-transgenic Cells

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,烟台264025

出　　处：《中国细胞生物学学报》2010年第6期873-878,共6页Chinese Journal of Cell Biology

基　　金：山东省自然科学基金(No.ZR2010CM061);烟台市科技计划项目(No.2008152);教育部留学回国人员科研启动基金项目(No.2007[1108]);鲁东大学科研基金项目(No.LZ20083305)资助项目~~

摘　　要：双载体转凝血Ⅷ因子基因(FⅧ)可有效克服腺相关病毒(AAV)载体容量限制,但FⅧ重链分泌的低效性导致重、轻链分泌的不均衡。重链分泌的低效性源自其A1区存在与内质网蛋白质分子伴侣结合的位点。本文在我们最近运用蛋白质剪接的双载体共转B区缺失型FⅧ(BDD-FⅧ)重链和轻链基因研究的基础上,将重链的A1区替换为猪FⅧ的A1区,用融合蛋白内含子的重链和轻链转基因实验,定量分析了重链的分泌及其对共转重链和轻链基因细胞分泌剪接BDD-FⅧ蛋白和活性的影响。结果显示,变构体重链单独转基因时其分泌得到明显改善,达到89±12 ng/ml,明显高于人BDD-FⅧ重链的分泌(25±9 ng/ml);该变构体重链与轻链共转基因细胞分泌的剪接变构体BDD-FⅧ和活性分别为219±51 ng/ml和1.47±0.22 U/ml,明显高于剪接的人BDD-FⅧ的分泌量和活性(1 16±32 ng/ml和0.8±0.11 U/ml)。单独变构体重链和轻链转基因细胞合并培养后,其培养上清中检测到剪接的变构体BDD-FⅧ和活性,分别为38±7 ng/ml和0.22±0.05 U/ml,提示为不依赖细胞机制的蛋白质剪接所产生。结果表明,A1区替换后重链分泌的增强,可促进基于蛋白质剪接技术的双载体共转重链和轻链基因细胞分泌的剪接BDD-FⅧ水平和活性,并可缓解链分泌的不均衡性,为动物体内应用双AAV载体共转BDD-FⅧ重链和轻链基因研究奠定了实验基础。Dual-vector delivery of coagulation factorⅧ（FⅧ） gene is an alternative strategy to overcome package limitation of adeno-associated virus（AAV） vectors,but the main drawback is chain imbalance due to the inefficient secretion of the heavy chain.It demonstrated that the inefficient heavy chain secretion is caused by binding of its A1 domain to chaperon protein of ER.Our recent work showed that protein splicing could be used for a dual-vector co-delivery of a B-domain-deleted FⅧ（BDD-FⅧ） heavy and light chain genes. Currently,we observed the secretion of A1 domain substituted human BDD-FⅧheavy chain by A1-domain of porcine FⅧand its effect on secretion of spliced BDD-FⅧand corresponding activity by co-transfection of an intein-fused heavy and light chain genes.The results showed that the secretion of variant heavy chain by itself displays an obvious enhancement in terms of secretion compared to the wild-type heavy chain（89±12 ng/ml vs 25±9 ng/ml）.The amount of spliced variant BDD-FⅧand activity in the culture supernatants of heavy and light chain co-transgenic cells were 219±51 ng/ml and 1.47±0.22 U/ml,respectively,higher than those of human BDD-FⅧheavy and light chain co-transgenic cells（116±32 ng/ml and 0.8±0.11 U/ml）.The spliced variant BDD-FⅧand activity were also detected in the culture supernatant of combined cells separately transfected with variant heavy and light chain genes（38±7 ng/ml and 0.22±0.05 U/ml） indicating the cellular mechanism-independent protein splicing.It suggests that enhanced secretion of the A1-domain substituted heavy chain could improve secretion of spliced BDD-FⅧand activity and relieve the chain imbalance secreted by the heavy and light chain co-transgenic cells with a protein splicing-based dual-vector.It provided evidence for ongoing research for an in vivo BDD-FⅧheavy and light genes co-transfer by a dual-AAV vector in animal models.

关 键 词：凝血Ⅷ因子 变构体重链 分泌 蛋白内含子 蛋白质剪接 

分 类 号：R554.1[医药卫生—血液循环系统疾病]