人GM-CSF基因的克隆及其稳定表达细胞系的建立

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

目的研究和建立人粒细胞-巨噬细胞集落刺激因子(gramulocyte/macrophase colony-stimulatingfactor,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Westernblotting、ELISA及其生物活性测定方法。结果将扩增出的GM-CSF cDNA克隆到pcDNA 3.1 A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850 ng/106细胞。结论本文建立的细胞系能够高效表达具有生物学活性的重组人GM-CSF。

Objective Establish a stable cell line to produce human granulocyte-macrophage colony-stimulating factor（hGM-CSF） with high level expression in order to reduce production cost. Methods Amplify 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. Results Human 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, follow;rig by G418 selection. The gene product was identified to have biological activity. The average yield of hGM-CSF was 850 ng/106 cell. Conclusion The established cell line can highly-productive express of hGM-CSF with biological activity.