人凝血因子_cDNA体外真核表达的初步研究.pdfVIP

4·64 · Chin J Hematol ,September 1998 ,Vol 19 ,No. 9 人凝血因子 ⅧcDNA 体外 真核表达的初步研究 诸江 　王鸿利 　于立志 　王学锋 　郭为民　陈赛娟 　戚正武 　王振义 　陈竺 　　摘要 　目的:建立人凝血因子 ⅧcDNA 体外真核高效表达的体系 。方法 :将人 F ⅧB 区大部分缺失 (Δ ) aa7601639 的 F ⅧcDNA 插入pCI 真核表达载体 、p GRE5. 2/ EBV 载体和逆转录病毒载体 pMSCV ,构 建了 5 种表达框架 ,在体外转染和感染了多种靶细胞 。结果 :pCI Ⅷ在 N IH3 T3 细胞产生了低水平表 达 ,而p GRE5. 2/ EBV Ⅷ和 pMSCV Ⅷ系列分别在 Hela 细胞和 Bosc23 逆转录病毒包装细胞中呈较高水 平的表达 。Bosc23 细胞培养上清感染的N IH3 T3 和 32DC 不能有效地产生 F Ⅷ。结论 :在 F Ⅷ体外表达 及基因治疗的方案设计中 ,靶细胞的选择是一重要因素 。 关键词 　凝血因子 Ⅷ　　基因表达 A preliminary study on the in vitro eukaryotic expression of human Factor ⅧcDNA 　Zhu Jiang , W ang Hongli , Y u L iz hi , et al . S hanghai Institute of Hem atology , R uijin Hospital , S hanghai Second Medical U niversity , S hanghai 　200025 　　Abstract 　Objective : To evaluate the expression efficiency of a cDNA sequence of human clotting factor ( ) Ⅷ 4. 7kb , B domaindeleted in in vitro systems. Methods : After insertion of the cDNA into several mam malian expression vectors , such as retroviral vector pMSCV , EB virusbased vector p GRE5. 2/ EBV and eu karyotic expression plasmid pCI , the expression of these constructs were tested in a variety of cells. Results : All the three kinds of constructspCI Ⅷ,p GRE5. 2/ EBV Ⅷand pMSCV Ⅷwere able to direct F Ⅷsynthesis in N IH3 T3 , Hela and Bosc23 cells , respectively , while the pMSCV Ⅷand p GRE5. 2/ EBV Ⅷproduced rela tively high levels of F Ⅷactivity (up to 0. 7 U/ ml and 2. 0 U/ ml from 24h to 48h , respectively , after trans fection with lipofectamine) . The three forms of pMSCV Ⅷ vector worked in a similar efficacy in Bosc 23 cells , but this function was not detected in N IH3 T3 , ψ p and GP + E86 as well as 32