Production of Human Interferons by Mouse Cells

在这个项目中，我们开发了哺乳动物细胞系统，利用重组DNA技术产生人干扰素（IFN）和粒细胞集落刺激因子（G-CSF）。已分别从Con A刺激的人脾细胞和产生G-CSF的人鳞癌细胞（CHU-2）中克隆出人ifn和G-CSF的cdna。通过克隆两种不同的G-CSF cdna，揭示了两种不同的G-CSF mRNA是由单一前体mRNA的选择性剪接产生的。将人IFNs或G-CSF的cdna置于SV40早期启动子的启动子下，并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）杂交质粒转化小鼠C127 I细胞，转化后的细胞能组成性地分泌人干扰素或G-CSF。部分转化细胞能高效地产生IFNs或G-CSF， IFNs的分泌速率为1.0 mg /l， G-CSF的分泌速率为10- 20mg /l。人IFN- < γ >和G-CSF已从转化细胞的培养基中纯化至均匀性。小鼠细胞产生的IFN- < γ >和G-CSF被糖基化，这些蛋白的nh端氨基酸序列与天然蛋白相同，表明这两种蛋白都被正确加工。重组人G-CSF皮下注射小鼠后，观察到明显刺激颗粒生成和脾肿大。

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.