人GM-CSF基因的克隆及其稳定表达细胞系的建立

目的研究和建立人粒细胞-巨噬细胞集落刺激因子（gramulocyte／macrophase colony-stimulating factor，GM—CSF）造血生长因子的高效表达细胞系，以降低生产成本。方法GM—CSF cDNA基因的扩增采用RT—PCR方法；基因转移采用电转移方法；细胞系采用小鼠B淋巴细胞系（L1／2）；表达产物鉴定采用Western blotting、ELISA及其生物活性测定方法。结果将扩增出的GM-CSF cDNA克隆到pcDNA3．1A载体上，构建成GM—CSF基因的重组表达载体（pcDNA—GMCSF）；经电转移将GM—CSF cDNA转移L1／2细胞中，通过G418的筛选，得到稳定表达重组人GM—CSF的细胞系。经过分析证明表达的重组人GM-CSF具有生物学活性，平均表达水平可达850ng／10^6细胞。结论本文建吐的细胞系能够高效表达具有生物学活性的重组人GM—CSF。

CLONNING OF HUMAN GM-CSF GENE AND ESTABLISHMENT OF ITS STABLE EXPRESSION CELL LINE

ObjectiveEstablish a stable cell line to produce human granulocyte-macrophage colony-stimulating factor(hGM-CSF) with high level expression in order to reduce production cost.MethodsAmplify GM-CSF cDNA with RT-PCR method;Transfect gene into L1/2 cell line by electroporation;Identify gene products by Western Blotting,ELISA and biological assay.ResultsHuman GM-CSF cDNA expression vector was prepared by inserting the hGM-CSF cDNA into pcDNA3.1A.Stable cell line expressing human GM-CSF was established by transfecting L1/2 cells with electroporation,following by G418 selection.The gene product was identified to have biological activity.The average yield of hGM-CSF was 850 ng/10~6 cell.ConclusionThe established cell line can highly-productive express of hGM-CSF with biological activity.