EFFICIENT CONSTITUTIVE PRODUCTION OF HUMAN IFN-GAMMA IN CHINESE-HAMSTER OVARY CELLS
EFFICIENT CONSTITUTIVE PRODUCTION OF HUMAN IFN-GAMMA IN CHINESE-HAMSTER OVARY CELLS
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DOI：
10.1089/dna.1986.5.181
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发表时间：
1986-06-01
期刊：
影响因子：
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通讯作者：
REVEL, M
中科院分区：
文献类型：
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作者：
MORY, Y;BENBARAK, J;REVEL, M
一段5.6kb的人类基因组DNA片段，包含免疫干扰素-γ的整个基因。通过其5‘端非翻译区与猴病毒40(SV40)T抗原基因的相应区域融合。所用的SV40早期启动子含有一个93-bp重复序列的转录增强子元件。以SV40-二氢叶酸基因为选择标记，将超螺旋DNA导入中国仓鼠卵巢(CHO)细胞。干扰素-γ的结构性表达。原代转化子中的基因是高表达的，特别是如果除了SV40启动子外还存在哈维小鼠肉瘤病毒长末端重复序列(LTR)。甲氨蝶呤选择基因扩增后，CHO-γ。获得了产生150-200万个单位的干扰素-γ的细胞系。每百万个细胞和每天(每个细胞每分钟200,000个分子)。代谢标记显示，在人干扰素-γ中，这种细胞分泌的蛋白质有90%以上。对单抗进行一步免疫亲和层析，得到纯的干扰素-γ。1-2倍。108单位/毫克蛋白质。就像干扰素-伽马。来自人类白血球的干扰素-γ。来自Cho-.Gamma。细胞是26,000和20,000道尔顿的两种糖蛋白的混合物，带有微量的未糖基化的17,000道尔顿多肽。在1%的血清中大规模培养，通常每天产生超过60万个单位的人干扰素-γ/毫升培养。
A human genomic DNA segment of 5.6 kb containing the entire gene for immune interferon-.gamma. was fused through its 5''-untranslated region to the corresponding region of the simian virus 40 (SV40) T-antigen gene. The SV40 early promoter used contained a modified transcriptional enhancer element with a 93-bp repeat. Supercoiled plasmid DNA was used to transfect Chinese hamster ovary (CHO) cells, the selectable marker being a SV40-dihydrofolate gene construct. Constitutive expression of the IFN-.gamma. gene in primary transformants was high, especially if a Harvey murine sarcoma virus long terminal repeat (LTR) was present in addition to the SV40 promoter. After gene amplification by methotrexate selection, CHO-.gamma. cell lines were obtained that produce 1.5-2 million units of IFN-.gamma. per million cells and per day (200,000 molecules per cell per minute). Metabolic labeling showed that over 90% of the protein secreted by such cells in human IFn-.gamma.. A one-step immuno-affinity chromatography on monoclonal antibodies yielded pure IFN-.gamma. with 1-2 .times. 108 units/mg protein. Like IFN-.gamma. from human white blood cells, the IFN-.gamma. from CHO-.gamma. cells is a mixture of two glycoproteins of 26,000 and 20,000 daltons with traces of the unglycosylated 17,000-dalton polypeptide. Large-scale cultures in 1% serum routinely yield over 600,000 units of human IFN-.gamma./ml culture per day.