Production of Human Interferons by Mouse Cells

在本项目中，我们利用重组DNA技术建立了产生人干扰素(IFN)和粒细胞集落刺激因子(G-CSF)的哺乳动物细胞系统，分别从ConA刺激的人脾细胞和产生G-CSF的人鳞癌细胞(CHU-2)中克隆了人IFN和G-CSF的cDNA。通过克隆两个不同的G-CSFcDNAs，发现两个不同的G-CSFmRNAs是由单个前体mRNA的选择性剪接而产生的。将人IFN或G-CSF基因克隆到SV40早期启动子的启动子下，以牛乳头瘤病毒为载体导入小鼠C127 I细胞。用牛乳头瘤病毒-干扰素(G-CSF)杂合载体转化小鼠C127 I细胞，转化后的细胞可分泌人干扰素或G-CSF。部分转化细胞能高效产生IFN或G-CSF1.0 mg/L或10-20 mg/L。人干扰素和粒细胞集落刺激因子已从转化细胞条件培养上清液中纯化至均一。对小鼠细胞产生的干扰素、γ-GT和G-CSF进行糖基化，其NH-末端氨基酸序列与天然蛋白一致，说明两种蛋白加工正确。重组人粒细胞集落刺激因子皮下注射对小鼠粒系生成和脾肿大有明显的刺激作用。

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.