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Production of Human Interferons by Mouse Cells

小鼠细胞产生人干扰素

基本信息

批准号：
59870010
负责人：
NAGATA Shigekazu
金额：
$ 5.38万
依托单位：
Institute of Medical Science, University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1984
资助国家：
日本
起止时间：
1984 至 1986
项目状态：
已结题

项目摘要

In this project, we have developed the mammalian cell system to produce human interferon ( IFN ) and granulocyte colony-stimulating factor ( G-CSF ) using the recombinant DNA technology.The cDNAs for human IFNs and G-CSF have been cloned from the Con A- stimulated human spleen cells or human squamous carcinoma cells ( CHU-2 ) producing G-CSF, respectively. By cloning of two different G-CSF cDNAs, it was revealed that two different G-CSF mRNAs are produced by the alternative splicing from the single precursor mRNA. The cDNAs for human IFNs or G-CSF were placed under the promoter of SV40 early promoter and introduced into mouse C127 I cells using bovine papilloma virus as a vector. The mouse C127 I cells were transformed by bovine papilloma virus-IFN ( G-CSF ) hybrid plasmid, and the transformed cells could secrete human IFNs or G-CSF, constitutively. Some of the transformed cells could produce IFNs or G-CSF very efficiently at the rate of 1.0 mg / l for IFNs or 10-20 mg /l for G-CSF. Human IFN- <gamma> and G-CSF have been purified to homogeneity from the medium conditioned with the transformed cells. IFN- <gamma> and G-CSF produced by mouse cells were glycosylated, and the NH-terminal amino acid sequence of those proteins were identical to that of the native proteins, indicating both proteins were processed correctly. When the recombinant human G-CSF was subcutasneously administrated into mice, a remarkable stimulation of granulopoiesis and splenomegaly was observed.

在这个项目中，我们开发了利用重组DNA技术生产人干扰素（IFN）和粒细胞集落刺激因子（G-CSF）的哺乳动物细胞系统。人IFN和G-CSF的cDNA分别从Con A刺激的人脾细胞或人鳞状癌细胞（CHU-2）产生G-CSF中克隆。通过克隆两种不同的 G-CSF cDNA，发现两种不同的 G-CSF mRNA 是通过单个前体 mRNA 的选择性剪接产生的。将人IFN或G-CSF的cDNA置于SV40早期启动子的启动子下，并使用牛乳头瘤病毒作为载体导入小鼠C127I细胞中。牛乳头瘤病毒-干扰素(G-CSF)杂合质粒转化小鼠C127 I细胞，转化细胞可组成型分泌人干扰素或G-CSF。一些转化的细胞可以非常有效地产生IFN或G-CSF，IFN的产量为1.0mg/l，G-CSF的产量为10-20mg/l。人IFN-γ和G-CSF已从用转化细胞条件化的培养基中纯化至同质。小鼠细胞产生的IFN-γ和G-CSF被糖基化，并且这些蛋白质的NH末端氨基酸序列与天然蛋白质的相同，表明这两种蛋白质都被正确加工。当重组人G-CSF皮下给予小鼠时，观察到显着刺激粒细胞生成和脾肿大。