转粒细胞-巨噬细胞集落刺激因子基因真核表达质粒的构建及生物学活性鉴定

Construction of granulocyte-macrophage colony stimulating factor gene eukaryotic expressing plasmid and identification of its biological activity

目的 构建小鼠转粒细胞 巨噬细胞集落刺激因子 (mGM CSF)真核表达质粒 pcDNA3 GM CSF ,转染红白血病细胞系FBL 3,并鉴定其活性。方法 采用分子克隆技术 ,构建mGM CSF真核表达质粒 pcDNA3 GM CSF ,电穿孔法将其导入红白血病细胞系FBL 3,G4 18筛选G4 18抗性细胞 ,限制性稀释法筛选G4 18抗性细胞克隆。PCR和RT PCR方法鉴定GM CSF基因整合和稳定表达。骨髓造血祖细胞增殖实验和骨髓造血祖细胞集落刺激实验鉴定其生物学活性。结果 采用PCR方法扩增mGM CSFcDNA序列 ,BamHⅠ和EcoRⅠ双酶切后装入 pcDNA3质粒 ,构建真核表达质粒 pcDNA3 GM CSF ,酶切和测序结果与预期相符 ,无插入、丢失、突变 ,方向正确。PCR和RT PCR结果显示 ,GM CSF基因整合到受体细胞染色体中并稳定表达。表达GM CSF的FBL 3 GM CSF细胞培养上清可明显刺激小鼠骨髓单个核细胞增殖 ,并能刺激干细胞形成克隆 ,形成克隆数为 (5 4 .6 7± 4 .83)个 ,形成率为 0 .5 4 7%。结论 构建mGM CSF真核表达质粒pcDNA3 GM CSF ,获得稳定表达该基因并具有生物学活性的细胞克隆 ,为制备转GM CSF基因瘤苗 。

Objective To construct mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) gene eukaryotic expressing plasmid pcDNA3-GM-CSF, to transfect the recombinant into erythroleukemia cell line FBL-3, and identify their biological activity.Methods GM-CSF gene eukaryotic expressing plasmid was constructed by subclone and recombinant was transfected into FBL-3 cells by electroporation. After screening by G418 and cloning by limiting dilution,we obtained positive cell clones(FBL-3-GM-CSF). PCR and RT-PCR were used to identify the integration and stable expression of GM-SF gene in FBL-3-GM-CSF cells. The biological activity was confirmed by the hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay. Results Mouse GM-CSF cDNA was amplified from the prokaryotic expressing plasmid PET-30a(+)-GM-CSF by PCR firstly and BamH Ⅰ and EcoRⅠrestriction sites were introduced. The inserted fragment was cut by BamH Ⅰ and EcoR Ⅰ digestion and ligated into pcDNA3 vector. The pcDNA3-GM-CSF eukaryotic expressing plasmid was constructed. The recombinant was cleared with appropriate endoneucleases and sequenced. The findings showed that the orientation of the insert was correct, while no rearrangement or mutation was found. PCR and RT-PCR assay showed that GM-CSF gene had integrated into FBL-3-GM-CSF cells and stably expressed. The hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay demonstrated that the cultured supernatant of FBL-3-GM-CSF cells of expressing GM-CSF should obviously stimulate proliferation of murine marrow mononuclear cells, and could stimulate hematopoietic progenitor cell colony formation. The number of colony formation was 54.67±4.83. The rate of colony formation was 0.547 %.Conclusions GM-CSF gene eukaryotic expressing plasmid is constructed successfully. A cell clone, which can express stably GM-CSF gene and possess biological activity,is obtained. Our studies have founded the base for the preparation of GM-CSF gene-modified vaccine of tumor cell and the study of feasibility of immune therapy of leukemia.