【摘要】 目的研究链亲和素标记的人肿瘤坏死因子α(SA-TNF-α)融合蛋白的纯化、复性方法,并研究其生物学功能。方法在大肠杆菌中表达SA-TNF-α融合蛋白,对表达的SA-TNF-α融合蛋白采用镍金属螯合(Ni-NTA)层析柱进行纯化,分别在尿素体系和盐酸胍体系中复性,Western blot对其进行鉴定。MTT法检测SA-TNF-α融合蛋白对L929细胞的杀伤活性,流式细胞仪分析SA-TNF-α融合蛋白对生物素化的MB49细胞锚定修饰率。结果SA-TNF-α融合蛋白在大肠杆菌中实现了高效表达,表达的目标蛋白占菌体蛋白30%以上,镍金属螯合(Ni-NTA)层析柱纯化的SA-TNF-α融合蛋白纯度达到95.7%,经过两种体系复性形成的SA-TNF-α融合蛋白的二聚体和多聚体均具有双功能活性:既对L929细胞有杀伤活性又能锚定修饰生物素化的MB49细胞,锚定修饰率大于90%。结论初步建立了SA-TNF-α融合蛋白制备工艺,SA-TNF-α二聚体和多聚体均具有双功能活性,SA-TNF-α融合蛋白的研制有望为肿瘤的治疗提供新的治疗方法及新型药物。更多还原

【Abstract】 Objective To study the purification,refolding and bioactivity of streptavidin-tagged human tumor necrosis factor-α(SA-TNF-α) bi-functional fusion protein.Methods SA-TNF-α fusion protein was expressed in BL21(DE3) host bacteria,purified using Ni-NTA affinity chromatography and refolded by dilution and dialysis followed by identification using Western blotting.The effect of SA-TNF-α fusion protein against L929 cells was evaluated by MTT assay.Flow cytometry was used to analyze the surface modification of biotinylated MB49 tumor cells by SA-TNF-α fusion protein.Results Recombinant SA-TNF-α fusion protein was expressed in BL21(DE3) at about 30% of total bacterial protein,with a purity of about 95% after purification.The SA-TNF-α fusion protein existed as dimmers,tetramers and higher order structures after refolding.The fusion protein exhibited a bi-functionality by inhibiting L929 cells and SA-mediated high-affinity binding to biotinylated cell surfaces,with an anchor modification rate of above 90%.Conclusion The dimmers,tetramers and higher order structures of the obtained SA-TNF-α fusion protein all exhibit a bi-functionality,and may serve as a potential candidate therapeutic agent for tumors.更多还原