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，其翻译后修饰对治疗效果很重要。促红细胞生成素（EPO）是一种糖蛋白，是其中的代表。氧在动物细胞培养中是一种限制性营养物质，为了高效生产有用蛋白质，氧的供应仍有待改进。在哺乳动物细胞中，已经发现了许多诱导缺氧的基因。诱导这些主要是由于转录激活因子，缺氧诱导因子-1 （HIF-1）与缺氧反应增强子（HRE）结合而激活转录。在本项目中，我们利用多种启动子和HRE，以及EPOcDNA作为报告基因，开发了在常氧或缺氧条件下都能高效产生重组蛋白的动物细胞培养。利用中国仓鼠卵巢（CHO）细胞，我们发现在缺氧条件下，HRE不仅可以激活EPO基因的启动子，还可以激活CMV和ef -1 α基因的启动子，这两种基因在动物细胞中都非常活跃。LDHA基因也是可缺氧的。我们制备了含有EPO cDNA转录受HRE控制的质粒和LDHA基因启动子的CHO克隆。我们研究了CHO细胞对EPO的氧依赖性生产，并比较了不同氧浓度下EPO生产的特征，提出了一种新的重组蛋白生产方法，该方法可以不受氧供应的困扰。

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.