链亲和素标记的人粒细胞巨噬细胞-集落刺激因子融合蛋白的制备

摘要

Objective To obtain streptavidin-tagged human granulocyte-macrophage colony-stimulating factor (SA/hGM-CSF)fusion protein and evaluate its bioactivity.Methods PET24a-6His-SA-L-hGM-CSF and PET24a-hGM-CSF-L-SA-6His plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate the fusion proteins.The two fusion proteins were refolded by gradient dialysis after Ni-NTA affinity chromatography and finally purified using DEAE-sepharose FF anion exchange chromatography.MTT method was used to evaluate the effect of SA/hGM-CSF fusion proteins in inducing the proliferation of human erythroleukemia cells (TF-1).The efficiency of the fusion proteins for surface modification of biotinylated MB49 tumor cells was evaluated by flow cytometry.Results The recombinant fusion proteins SA-hGM-CSF and hGM-CSF-SA were highly expressed in Rosetta (DE3) at about 20％ of the total bacterial proteins,with a purity of about 96％ after purification.The two fusion proteins exhibited bifunctional activities,namely the pro-proliferation effect on human erythroleukemia cells (TF-1) and SA-mediated high-affinity binding to biotinylated cell surfaces (with an anchoring modified rate of about 99％ ).Conclusion SA/hGM-CSF bi-fusion proteins obtained in this study lays the groundwork for the development of cancer cell vaccines with surface modification by hGM-CSF.%目的 制备链亲和素(SA)标记的人粒细胞巨噬细胞-集落刺激因子(hGM-CSF)融合蛋白SA-hGM-CSF和hGM-CSF-SA,并鉴定其生物学活性.方法 构建原核表达质粒PET24a-6His-SA-L-hGM-CSF和PET24a-hGM-CSF-L-SA-6His,转化大肠杆菌Rosetta(DE3),用镍金属螯合(Ni-NTA)层析柱进行纯化,并对其进行复性.复性后蛋白用DEAE-Sepharose FF阴离子交换层析进一步纯化.MTT法检测融合蛋白对人红白血病细胞(TF-1)的增殖活性.流式细胞仪分析融合蛋白对生物素化的MB49细胞锚定修饰效率.结果 SA-hGM-CSF和hGM-CSF-SA两种融合蛋白在大肠杆菌中实现了高效表达,目标蛋白占菌体蛋白的20％以上,纯化后纯度达到96％以上.两种融合蛋白均具有双功能活性,即同时具有hGM-CSF的促进人红白血病细胞增殖的活性和SA介导的高效结合至表面生物素化的MB49细胞的功能(锚定修饰率大于99％).结论 研制的SA/hGM-CSF融合蛋白具有双重活性,可为研制hGM-CSF表面修饰的新型肿瘤细胞疫苗提供基础.