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(英文)  

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

作　　者：白莉[1] 胡志明[1] 王菲[1] 许晓玲[2] 夏昶[2] 金丽琴[2] 李金龙[1] 高基民[2] 

机构地区：[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　　处：《南方医科大学学报》2012年第10期1389-1393,共5页Journal of Southern Medical University

基　　金：Supported by National 863 Program(2006AA02Z4C4);SciTech Research Project of Baiyun District,Guangzhou(2009-SZ-40)~~

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。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.

关 键 词：人粒细胞-巨噬细胞集落刺激因子 链亲和素 肿瘤细胞疫苗 融合蛋白 

分 类 号：R363[医药卫生—病理学]