Expression and characterization of the product of a human immune interferon cDNA gene in Chinese hamster ovary cells.
Expression and characterization of the product of a human immune interferon cDNA gene in Chinese hamster ovary cells.
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人免疫干扰素cDNA基因在中国仓鼠卵巢细胞中的表达及产物特性。
DOI：
10.1073/pnas.80.15.4654
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发表时间：
1983
影响因子：
11.1
通讯作者：
W. Fiers
中科院分区：
文献类型：
--
作者：
Shaun J. Scahill;Rene Devos;Jose Van;Der;Heyden;W. Fiers
用编码人免疫干扰素的pSV2-干扰素-γ和编码小鼠二氢叶酸还原酶的两个质粒[pAdD26SV(A)-3]共转化中国仓鼠卵巢(CHO)细胞系，建立了高水平分泌人干扰素-γ的中国仓鼠卵巢细胞系。转化的CHO细胞株E-10B和E-10C产生的干扰素-γ可达约50000单位/毫升的培养液，与刺激的淋巴细胞相比是有利的。此外，由于这些研究中使用的人干扰素-γ基因是在猿猴病毒40早期启动子的转录控制下进行的，因此人干扰素-γ的产生是结构性的，因此不需要诱导。CHO产生的人干扰素-γ在NaDodSO4/聚丙烯酰胺凝胶上以两条带的形式迁移，分子量分别为25,000和21,000。这两个物种被证明是单一基因的产物。由于天然人干扰素-γ的相对分子质量约为55000，因此很可能是二聚体。我们已经证明，这种二聚体的亚基不能通过二硫键连接(S)。CHO细胞中的HU干扰素-γ很可能是糖基化的，这现在应该可以在动物实验中比较糖基化和非糖化(细菌产生)的HU-干扰素-伽马的生物学活性。
Cotransformation with two plasmids, one [pSV2-IFN-gamma] encoding human immune interferon (Hu IFN-gamma) and the other [pAdD26SV(A)-3] encoding mouse dihydrofolate reductase, has been used to establish Chinese hamster ovary (CHO) cell lines that secrete high levels of Hu IFN-gamma. Hu IFN-gamma production by the transformed CHO cell lines E-10B and E-10C reached approximately 50,000 units/ml of culture medium, which compares favorably with that of stimulated lymphocytes. Furthermore, as the Hu IFN-gamma cDNA gene used in these studies is under the transcriptional control of the simian virus 40 early promoter, Hu IFN-gamma production is constitutive and thus does not require induction. CHO-produced Hu IFN-gamma migrates as two bands corresponding to molecular weights of 25,000 and 21,000 on NaDodSO4/polyacrylamide gels. These two species are shown to be the products of a single gene. As the molecular weight of native Hu IFN-gamma is around 55,000, it is likely to be a dimer. We have shown that the subunits of such a dimer cannot be linked by a disulfide bridge(s). Hu IFN-gamma from CHO cells is likely to be glycosylated and this should now permit comparison of the biological activities of glycosylated and nonglycosylated (bacterially produced) Hu IFN-gamma in animal studies.