摘要
运用Flp InTM 定点整合表达系统构建表达组织型纤溶酶原激活剂 (tissue type plasminogenactivator ,t PA)的CHO工程细胞株。将pcDNA5 /FRT载体和携带t PA基因的 pJOD S载体用Hind/KpnI双酶切 ,再将回收的 2 .0kbt PAcDNA片段和pcDNA5 /FRT载体连接 ,构建 pcDNA5 /FRT/t PA质粒 ;应用Flp InTM定点整合表达系统 ,将t PAcDNA定点插入Flp lnTM CHO细胞基因组中的 1个单拷贝位点上 ,以潮霉素B筛选定点整合t PAcDNA的细胞克隆。经筛选和体外纤维蛋白溶解活性检测 ,获得了t PA表达水平为 15 0 0～ 2 0 0 0IU/ (10 6cells·day)的CHO工程细胞株。
To develop recombinant CHO cell line expressing t-P A via site-specific integration expression system. Both plasmid pcDNA5/FRT and pl asmid pJOD-S carried t-PA cDNA were enzymatically digested by Hind/Kpn I, and the expressing plasmid pcDNA5/FRT/t-PA was constructed by inserting an 2.0 kb t-PA cDNA into pcDNA5/FRT. The recombinant CHO cell line expressing t-P A was constructed by using Flp-In TM site specific integration expression system and selected by Hygromycin B. After two round of selection, ten clones re combinant CHO cell with the capacity of expressing t-PA at 1 500～ 2 000 IU/(10 6cells·day) were obtained. The CHO expression system based o n Flp-In TM site specific integration could be used for the efficient expression of foreign gene.
出处
《药物生物技术》
CAS
CSCD
2004年第4期221-224,共4页
Pharmaceutical Biotechnology
基金
国家高技术研究与发展计划项目资助 (No .2 0 0 1AA2 15 461)
