Tsuchiya M, Nomura H, Asano S, Kaziro Y, Nagata S
EMBO J. 1987 Mar;6(3):611-6. doi: 10.1002/j.1460-2075.1987.tb04798.x.
Mouse C127I cells were transformed with a chimeric plasmid consisting of bovine papillomavirus DNA and human granulocyte-colony-stimulating factor (G-CSF) cDNA placed under the control of the SV40 early promoter. The transformed cells secreted constitutively a high level of human G-CSF, 10-20 micrograms/ml in a low-serum medium. The secreted G-CSF has been purified to homogeneity by a two-step procedure including gel filtration and hydrophobic column chromatography. The purified recombinant G-CSF runs as a single band with an apparent Mr of 19,000 on a polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This value corresponds to that of the native human G-CSF purified from the medium conditioned by human carcinoma CHU-2 cells. The recombinant human G-CSF was as active as native G-CSF in vitro in supporting proliferation of mouse NFS-60 cells and stimulating colony formation from human as well as mouse bone marrow cells. When the recombinant human G-CSF was subcutaneously administrated into mice, a remarkable stimulation of granulopoiesis and splenomegaly was observed.
用一种嵌合质粒转化小鼠C127I细胞，该嵌合质粒由牛乳头瘤病毒DNA和置于SV40早期启动子控制下的人粒细胞集落刺激因子（G-CSF）cDNA组成。转化细胞在低血清培养基中持续分泌高水平的人G-CSF，浓度为10 - 20微克/毫升。分泌的G-CSF已通过两步法纯化至均一，该方法包括凝胶过滤和疏水柱层析。在十二烷基硫酸钠存在下进行的聚丙烯酰胺凝胶电泳中，纯化的重组G-CSF呈现为一条单一的条带，表观分子量为19,000。该值与从人癌CHU-2细胞条件培养基中纯化的天然人G-CSF的值一致。重组人G-CSF在体外支持小鼠NFS-60细胞增殖以及刺激人及小鼠骨髓细胞形成集落方面与天然G-CSF活性相同。当将重组人G-CSF皮下注射到小鼠体内时，观察到粒细胞生成受到显著刺激且脾脏肿大。
