High density culture of animal cells by the use of hypoxia-response enhancer

哺乳动物细胞用于生产重组蛋白，其翻译后修饰对于治疗功效是重要的。促红细胞生成素（EPO），一种糖蛋白，是其中的代表。氧是动物细胞培养中的限制性营养素，其供应仍值得改进以高效生产有用蛋白质。在哺乳动物细胞中发现了许多低氧诱导基因，它们的诱导主要是通过转录激活因子-低氧诱导因子-1（HIF-1）与低氧应答增强子（HRE）结合而激活转录。在本项目中，我们开发了动物细胞培养物，通过该动物细胞培养物可以在常氧或低氧条件下高效地生产重组蛋白，使用各种启动子和HRE，以及EPOcDNA作为报告基因。用中国仓鼠卵巢细胞（CHO）研究表明，在低氧条件下，HRE不仅能激活EPO基因的启动子，还能激活CMV和EF-1 α基因的启动子，这两个基因在动物细胞中都是非常活跃的。LDHA基因也是低氧诱导的。我们制备了含有EPO cDNA转录受HRE和LDHA基因启动子控制的质粒的CHO克隆。我们研究了CHO细胞的EPO的氧依赖性生产，并比较了在不同氧浓度下生产的EPO的特性，提出了一种新的生产重组蛋白的方法，通过该方法可以不受氧气供应的困扰。

Mammalian cells are used for producing recombinant proteins whose post-translational modifications are important for therapeutic efficacy. Erythropoietin (EPO), a glycoprotein, is a representative of these. Oxygen is a limiting nutrient in animal cell culture and its supply is still worthy of improvement for producing useful proteins with a high efficiency. In mammalian cells, a number of hypoxia-inducible genes have been found. Induction of these is mainly due to activation of transcription by binding of a transcriptional activator, a hypoxia-inducible factor-1 (HIF-1), to the hypoxia-response enhancer (HRE). In this project, we developed the animal cell culture by which recombinant proteins can be produced with a high efficiency under either normoxic or hypoxic conditions, using a variety of Promoters and HRE,and EPOcDNA as reporter. Using Chinese hamsterovary (CHO) cells, we show that, under hypoxic condition, the HRE can activate not only the promoter of EPO gene but also promoters of CMV and EF-1alpha genes, both of which are very active in animal cells. LDHA gene is also hypoxiainducible. We prepared CHO clones harboring the plasmid in which transcription of EPO cDNA is under control of HRE and the promoter of LDHA gene. We examined oxygen-dependent production of EPO by CHO cells and compares characteristics of EPO produced in various oxygen concentrations, proposing a new method for production of recombinant proteins by which one is allowed not to be obsessed with oxygen supply.