Cytotechnology. 1999 Sep;31(1-2):103-9. doi: 10.1023/A:1008048928053.
To construct a recombinant protein highly producing cell lines, we have previously developed the Oncogene Activated Production (OAP) system by using BHK-21 cells. Here we verified the availability of the OAP system in CHO cells. We firstly generated 'primed' ras amplified CHO cells, ras clone I, by introducing human c-Ha-ras oncogene into CHO cells. This ras clone I enables quick and easy establishment of recombinant protein hyper producing cell lines by introduction reporter gene of interest. Then we generated I13 by introducing human interleukin 6 (hIL-6) gene as a reporter gene, which showed enhanced productivity rate as compared to A7 established by conventional method. Furthermore, we found that hIL-6 production level of I13 was slightly improved by raising the CO(2) concentration from 5 to 8% possibly because of the enhanced growth rate. We further introduced the E1A oncogene, which has been shown to have a synergistic effect on the recombinant protein production of the ras-amplified BHK-21 cells, then evaluated the productivity. When culture in 5% CO(2) condition, only the slight effect can be seen. However when cultured in 8% CO(2) condition, not only cell number, but also productivity increased significantly, resulted in great augmentation of hIL-6 production, maximum production being 88.6 mug/ml/3 days. This study demonstrates that recombinant protein production level reached commercially desirable level by utilizing our OAP system in CHO cells and optimizing the culture condition.
为了构建高效生产重组蛋白的细胞系，我们之前利用 BHK-21 细胞开发了原癌基因激活生产（OAP）系统。在这里，我们验证了该 OAP 系统在 CHO 细胞中的可用性。我们首先通过将人 c-Ha-ras 癌基因导入 CHO 细胞中生成了“引发”ras 扩增 CHO 细胞 ras 克隆 I。通过导入感兴趣的报告基因，该 ras 克隆 I 可以快速、轻松地建立重组蛋白高产细胞系。然后，我们通过引入人白细胞介素 6（hIL-6）基因作为报告基因生成了 I13，与通过传统方法建立的 A7 相比，其生产效率得到了提高。此外，我们发现通过将 CO2 浓度从 5%提高到 8%，I13 的 hIL-6 产量略有提高，这可能是由于生长速度的提高。我们进一步引入了 E1A 癌基因，该基因已被证明对 ras 扩增 BHK-21 细胞的重组蛋白生产具有协同作用，然后评估了其生产效率。在 5%CO2 条件下培养时，只能看到轻微的效果。然而，在 8%CO2 条件下培养时，不仅细胞数量增加，而且生产效率也显著提高，导致 hIL-6 产量大幅增加，最大产量为 88.6 mug/ml/3 天。本研究表明，通过在 CHO 细胞中利用我们的 OAP 系统并优化培养条件，重组蛋白的生产水平达到了商业上期望的水平。
