摘要
为了构建含人凝血因子Ⅷ基因的重组腺病毒载体系统，首先构建了含ＦⅧ（Ｂ结构域缺失型）的载体质粒ｐＡｄ－ｈＦⅧ和插入内含子结构的ｐＡｄＩ－ｈＦⅧ，经脂质体介导，证明两质粒的目的基因均能在ＣＯＳ－７细胞中表达有凝血活性的ＦⅧ因子，其中经纯化的载体质粒ｐＡｄ－ｈＦⅧ与ｐＢＨＧ１０经脂质体介导，共转染至２９３包装细胞，经同源重组和Ｅ１蛋白反式互补，形成Ｅ１／Ｅ３缺失型重组腺病毒载体颗粒Ａｄ－ｈＦⅧ．ＰＣＲ检则到病理２９３培养液上清的病毒ＤＮＡ中具有目的基因扩增片段，证明病毒ＤＮＡ含有ＦⅧ基因．经扩增、纯化、滴度测定，用于感染离体细胞ＣＯＳ－７、Ａ５４９、Ｌ９２９、２９３．证实该病毒能在上述细胞中高效转移和表达目的基因，其表达量具有一定范围内的剂量依赖性．ＣＯＳ－７细胞中，ＭＯＩ＞１０００时，有细胞毒效应，表达量反而下降．经耳缘静脉注射Ａｄ－ｈＦⅧ至家兔后，１～４周能测到一定水平的ＦⅧ蛋白，１周内升至最高峰．免疫组化研究表明感染的离体细胞和家兔肝等组织均有ＦⅧ表达，其中肝脏表达最高．
Both recombinant plasmids pAd hFⅧ (without SV40 intron) and pAdI hFⅧ(with SV40 intron) were first constructed for generation of recombinant adenoviral vectors.The human factorⅧ gene within both recombinant plasmids can be functionally expressed in COS 7 cells.A recombinant replication defective adenoviral vector Ad hFⅧ was rescued in 293 packaging cells by co tranfection of both plasmids pAd hFⅧ and pBHG10 containing Ad5 genome with deletions of packaging signal E1 and E3 regions.The insert gene was identified in adenoviral vector DNA from cpe(cytophatic effect) 293 cells culture medium.After propagation,purification,and titration,the vector was used for infection in vitro and in vivo .The results demonstrated that functional hFⅧ proteins were detected in transfected COS 7,A549,L929,293 cells.The concentration of hFⅧ increased in a linear fashion with increases in MOI of Ad hFⅧ in COS 7 cells.But significant cellular toxicity was observed in a parallel with increases in MOI beyond 1 000.The hFⅧ proteins were also detected in plasma of rabbits injected with Ad hFⅧ and peaked up to 154 ng/ml.The positive hFⅧ proteins were observed in transfected cells and livers of rabbits injected with the vector by immunohistochemistry assay.These results represent a major step forward into development of gene therapy for treating hemophilia A.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
1999年第1期36-41,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金
