人尿激酶原在CHO细胞中的基因扩增与高效表达

本文通过基因共转染和基因共扩增方法,在SV40病毒晚期启动子控制下成功地在中国仓鼠卵巢细胞二氢叶酸还原酶缺陷株(CHO-DHFR~-)中高效表达了人尿激酶原,其表达水平在5×10~(-6) mol/L氨甲喋呤存在下可达2—3μg/10~6细胞/24 h。采用PMA诱导可将表达水平提高至3.5—4μg/10~6细胞/24 h。相应的Pro-UKcDNA在细胞基因组上整合的拷贝数为200—300拷贝/细胞。Western blot分析表明,表达出来的重组Pro-UK与天然Pro-UK具有相似的分子量:S_(2444)测活法证明重组Pro-UK具有体外酰胺水解活性。     

Gene Amplification and High-Level Expression of Human Pro-Urokinase cDNA in Chinese Hamster Ovary Cells

Human Pro-Urokinase (Pro-UK) is expressed in CHO-DHFR- cells at high efficiency by co-transfecting the mouse dhfr gene and Pro-UK cDNA under the control of the SV40 late promoter. After gene co-amplification, the product level could reach 2-3 μg/106 cells/24 h in the presence of 5×10-6 mol/ L MTX, and the levels can be further raised to 3. 5-4 μg/106 cells/24 h by PMA superinduction. The copy number of Pro-UK cDNA in the genomes of host cells is about 200-300 copies/cell. The Western blot analysis shows that the recombinant Pro-UK has similar molecular weight to its natural counterpart, and also the amidolytic activity of the product is determined by S-2444 assay.