人尿激酶原cDNA在中国仓鼠卵巢细胞(CHO)中高效表达研究  

High Level Expression of Human Prourokinase cDNA in Chinese Hamster Ovary (CHO) Cells

作　　者：程度胜[1] 俞炜源[1] 韩素文[1] 李秀珍[1] 李风知[1] 胡宝成[1] 方继明[1] 黄翠芬[1] 

机构地区：[1]军事医学科学院生物工程研究所

出　　处：《生物工程学报》1993年第3期204-209,共6页Chinese Journal of Biotechnology

基　　金：国家生物高技术“863”资助项目

摘　　要：通过构建高效表达载体,改进转染方法,与二氢叶酸还原酶(dhfr)基因共扩增等手段在CHO细胞内高效表达了人尿激酶原(Pro-UK)cDNA。首先将pro-UK cDNA插入到SRα启动子的下游,构建成表达质粒pMG10102,在COS-7细胞内进行暂时性表达,结果表明此启动子的表达水平比SV40早期启动子高约5倍。然后将质粒pMG10102和pSV2-dhfr线性化后用磷酸钙共沉淀法转染CHO-dhfr^-细胞,经一系列筛选后获得20个能表达pro-UK的细胞克隆,纤维蛋白溶解平板法(FAPA)测定表达水平为12.5—100IU/10~6 cells/d.再经MTX加压共扩增,得到9株高表达细胞系,其中最高的表达水平达到400—500IU/10~6 cells/d。经2—3个月连续传代,表达水平未下降,表明细胞株是稳定的。Western Blot分析证明细胞分泌的重组pro-UK具有与天然pro-UK相同的分子量,而且培养液中不加蛋白酶抑制剂时,分泌的重组UK大部分为单链(60%以上)。We have used Chinese Hamster Ovary (CHO) cells to express high levels of human prourokinase gene cDNA with recourse to construction of good expression vector, the improvement of transfection technique and gene coamplification. First, we constructed expression plasmid pMG10102 by placing Pro-UK cDNA under the control of SRa promoter/SV 40 polyadenylation signals and expressed it transiently in COS-7 cells. Expression level was about 5 folds higher comparison with SV40 early promoter. Linear plasmids pMG 10102 and pSV 2-dhfr were then cotransfected into CHO-dhfr^- cells by calcium phosphate coprecipitation and cells were cultured in selective medium. Twenty transformants expressing Pro-UK were picked,the range of expression levels was 12.5—100 IU/10~6 cells/day.When subjected to stepwise selection of methortrexate (MTX), stable cell lines were obtained that secreted up to 400—500 IU/10~6 cells/day.Western Blot analysis showed that molecular weight of secreted recombinant Pro-UK was the same as that of natural Pro-UK which is 52 kDa and more than 60 percent of expression production was single chain urokinase (rscUK) without protease inhibitor in medium.

关 键 词：尿激酶原 尿激酶原基因 CDNA 基因工程 高效表达 

分 类 号：R392－33[医药卫生—免疫学] R394.8[医药卫生—基础医学]