Generation of streptavidin-tagged human-granulocyte macrophage colony-stimulating factor fusion proteins

南方医科大学学报 ›› 2012, Vol. 32 ›› Issue (10): 1389-.

Generation of streptavidin-tagged human-granulocyte macrophage colony-stimulating factor fusion proteins

白莉，胡志明，王菲，许晓玲，夏昶，金丽琴，李金龙，高基民

1南方医科大学生物技术学院生物治疗研究所，广东广州510515；2温州医学院生命科学学院浙江省模式生物技术与应用重点实验室，浙江温州325035

出版日期:2012-10-20 发布日期:2012-10-20

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

1南方医科大学生物技术学院生物治疗研究所，广东广州510515；2温州医学院生命科学学院浙江省模式生物技术与应用重点实验室，浙江温州325035

Online:2012-10-20 Published:2012-10-20

摘要/Abstract

摘要： 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。

Abstract: Objective To obtain streptavidin-tagged human granulocyte-macrophage colony-stimulating factor (SA/hGM-CSF)
fusion protein and evaluate its bioactivity. MethodsPET24a-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 MB49tumor cells was evaluated by flow cytometry. ResultsThe recombinant fusion proteins SA-hGM-CSF and
hGM-CSF-SA were highly expressed in Rosetta (DE3) at about20% of the total bacterial proteins, with a purity of about96%
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% ).ConclusionSA/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.

白莉，胡志明，王菲，许晓玲，夏昶，金丽琴，李金龙，高基民. Generation of streptavidin-tagged human-granulocyte macrophage colony-stimulating factor fusion proteins[J]. 南方医科大学学报, 2012, 32(10): 1389-.

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