人粒系巨噬系克隆刺激因子基因导入真核细胞及其表达

目的：获得带糖链、近天然的重组人粒系巨噬系克隆刺激因子（ｈＧＭ－ＣＳＦ），以期为临床提供更安全、稳定的细胞因子。方法：采用基因重组技术，构建编码ｈＧＭ－ＣＳＦ的稳转载体ｐＭＥｎｅｏ－ｈＧＭ－ＣＳＦ，以酶切和猴肾细胞ＣＯＳ瞬时转染鉴定后，用磷酸钙－ＤＮＡ共沉淀法将重组质粒导入中国仓鼠卵细胞（ＣＨＯ）。结果：经Ｇ４１８加压筛选（８００μｇ／ｍｌ～１ｍｇ／ｍｌ），获得可表达ｈＧＭ－ＣＳＦ的细胞株。常规或载体培养ＣＨＯ－ｈＧＭ－ＣＳＦ细胞株上清可直接刺激ｈＧＭ－ＣＳＦ依赖细胞株的增殖，ＭＴＴ测定活性单位可达１×１０７ｕ／Ｌ原液。ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔ分析显示ｒｈＧＭ－ＣＳＦ主带在２８ＫＤ位左右，为２Ｎ糖链型。结论：此ＣＨＯ－ｈＧＭ－ＣＳＦ细胞株表达稳定，活性较高，具有开发价值。

Transfection of Human Granulocyte Macrophage Clone StimulatingFactor into Eucaryotic Cells and Its Expression

? Objective:The nearly natural recombinant human granuloctye macrophage clone stimulating factor with carbohydrate chain(hGM CSF) was obtained to provide more safe and stable cell factor than ever did for clinical application.Method:Using the gene recombination technique,there was structured the stably transfected trager pMEneo hGM CSF with code hGM CSF which was assayed by enzyme incision and by COS instan taneous transfection into monkey kidney cells,and then the recombinant plasmid was transfected into chinese hamster ovum (CHO) by calcium phosphate DNA co precipitation.Results:After screening under pressure by G418(800μg￣1mg/ml),the cell strain which could express hGM CSF was obtained.The supernatant fluid on which the CHO hGM CSF cell strain was cultured routinely or with carrier could dircetly stimulate prolifination of hGM CSF cell strain.The activity determined by MTT could reach 1×107U/L　(stock solution).The analysis performed by SDS PAGE and Western blot showed that the rhGM CSF main band was at the position of about 28 KD and was a 2N carbohydrate chain.Conclusion:This CHO hGM CSF cell strain is of stable expression,its activity is high,and it has high development value.