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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 prepare SA-hGM-CSF and hGM-CSF-SA labeled with streptavidin (SA) and to identify their biological activities. Methods The prokaryotic expression plasmids PET24a-6His-SA-L-hGM-CSF and PET24a-hGM-CSF-L-SA-6His were transformed into E.coli Rosetta (DE3) Purify and renature it. Refolded proteins were further purified by DEAE-Sepharose FF anion exchange chromatography. MTT assay was used to detect the proliferation of human erythroleukemia cells (TF-1). Flow cytometry analysis of fusion protein biotinylated MB49 cell anchoring modification efficiency. Results The fusion proteins of SA-hGM-CSF and hGM-CSF-SA were highly expressed in E. coli. The target protein accounted for more than 20% of the bacterial protein and the purity of the fusion protein was over 96% after purification. Both fusion proteins have bifunctional activity, that is, the activity of hGM-CSF that promotes the proliferation of human erythroleukemia cells and the function of SA-mediated efficient binding to surface biotinylated MB49 cells (anchoring modification rate greater than 99% ). Conclusions The developed SA / hGM-CSF fusion protein has dual activity and can provide a foundation for the development of a novel tumor cell vaccine with surface modification of hGM-CSF.