【摘要】 目的:制备链亲和素(SA)标记的鼠白细胞介素-15融合蛋白SA-mIL15和mIL15-SA,并研究其生物学功能。方法:构建pET24a-SA-L-mIL15和pET21a-mIL15-L-SA重组表达质粒,在大肠杆菌中表达融合蛋白SA-mIL15和mIL15-SA,对所表达的蛋白分别采用镍金属螯合(Ni-NTA)层析和阴离子交换(DEAE)层析进行纯化,透析复性。MTT法检测融合蛋白对ConA刺激的小鼠脾淋巴细胞增殖活性,流式细胞术(FCM)分析融合蛋白对生物素化的小鼠前列腺癌(RM-1)细胞表面锚定修饰效率。结果:SA-mIL15和mIL15-SA在大肠杆菌中实现了高效表达,约占细菌总蛋白的20%。制备的SA-mIL15和mIL15-SA融合蛋白纯度达到95%,并具有双重活性,即:mIL15促进ConA激活的小鼠脾淋巴细胞的增殖活性和SA介导的高效结合至表面已生物素化的RM-1细胞的功能(表面锚定修饰效率均大于95%)。其中,SA-mIL15双功能融合蛋白促进ConA激活的小鼠脾淋巴细胞增殖活性为1×106IU/mg,mIL15-SA活性为2×105IU/mg。结论:SA/mIL15双功能融合蛋白具有双重活性,为mIL15表面锚定修饰的肿瘤细胞疫苗的研制奠定了基础。更多还原

【Abstract】 AIM:To prepare and characterize streptavidin-tagged murine interleukin-15 fusion proteins.METHODS:pET24a-SA-L-mIL15 and pET21a-mIL15-L-SA plasmids were constructed and expressed in Rosetta(DE3) host bacteria to generate SA/mIL15 fusion proteins.SA-mIL15 fusion protein was purified through the Ni-NTA affinity chromatography,and mIL15-SA fusion protein through anion exchange chromatography,followed by refolding.The efficiency of surface modification of the fusion proteins on the biotinylated RM-1 tumor cells was evaluated by a flow cytometer.MTT method was used to evaluate the proliferating effect of SA/mIL15 fusion proteins on mouse spleen lymphocytes stimulated by ConA.RESULTS:Both SA-mIL15 and mIL15-SA fusion proteins were highly expressed in Rosetta(DE3) at about 20% of total bacterial proteins.They exhibited the bi-functionality:proliferation-promoting activity of mIL15 on mouse spleen lymphocytes with the specific activity of 1×106 IU/mg for SA-mIL15 or 2×105 IU/mg for mIL15-SA,and SA-mediated high-affinity binding to the biotinylated surfaces of RM-1 tumor cells with about 95% surface modification efficiency.CONCLUSION:SA/mIL15 bi-functional fusion proteins were generated,which made feasible the development of mIL15-surtface-modified cancer cell vaccine.更多还原