用于生产重组蛋白药物的抗凋亡CHO宿主细胞株的建立  

Construction of an Anti-apoptosis CHO Cell Line for Biopharmaceutical Production

作　　者：来大志[1] 付玲[1] 于长明[1] 齐连权[1] 翁少洁[1] 于婷[1] 王海涛[1] 陈薇[1] 

机构地区：[1]北京微生物流行病研究所,北京100071

出　　处：《生物工程学报》2003年第3期322-326,共5页Chinese Journal of Biotechnology

摘　　要：哺乳动物工程细胞在大规模培养生产重组蛋白时很容易发生细胞凋亡 ,从而导致生产过程提前终止 ,造成生产成本高昂。细胞代谢产物氨已被证明可以促进细胞凋亡 ,而线粒体膜整合蛋白Bcl 2可以通过促进线粒体膜完整性而抑制细胞凋亡。本实验应用谷氨酰胺合成酶加压系统在CHO工程细胞中高效表达中国仓鼠Bcl 2蛋白 ,使细胞具有抗凋亡能力的同时 ,利用谷氨酸和氨合成谷氨酰胺而有效降低培养基中氨的含量 。Mammalian cells are prone to apoptosis when cultured in large scale for production of biopharmaceuticals. And this will reduce production duration and result in high cost of production. Apoptosis is triggered by various factors, and delicately re gulated by a set of genes. Bcl 2, a component integrated in mitochondria membrane, is an important member of these genes. By maintaining the integrity of mitochondria membrane, Bcl 2 keeps cytochrome C from releasing into cytoplasm, and thus blocks the activation of caspases, and subsequent onset of apoptosis. Over expression of Bcl 2 has proven to be useful in blocking apoptosis in various cell lines, including CHO, hybridoma, myeloma, lymphoma and insect cells. Ammonia, a metabolite of cultured cells, however, showed apparent pro apoptosis activity. In living cells, ammonia can be utilized by glutamine synthetase (GS) to synthesize glutamine, and thus lower the concentration of ammonia in medium, and its negative effects. Glutamine is essential to living cells. If not added into medium, glutamine can only be synthesized by GS, which makes GS a qualified selection marker. This marker can be used for gene amplification by adding into medium increased concentration of MSX, an inhibitor of GS. In this study, we over expressed Bcl 2 using GS amplification in a recombinant CHO cell line stably expressing human interferon β. The modified cell line, with higher expression of Bcl 2 and lower production of ammonia, exhibited good anti apoptosis quality and higher interferon β production in continuous culture.

关 键 词：细胞凋亡 CHO细胞 谷氨酰胺合成酶 氨 重组蛋白药物 哺乳动物细胞 表达系统 

分 类 号：Q813[生物学—生物工程]