SA-hIL2双功能融合蛋白的研制及其生物学鉴定

Preparation and Characterization of SA-hIL2 Fusion Protein

目的:制备链亲和素标记的人白细胞介素-2(SA-hIL2)融合蛋白,并研究其生物学功能。方法:构建SA-L-IL2-pET24重组表达质粒,在大肠杆菌中表达SA-hIL2融合蛋白,对表达的SA-hIL2融合蛋白采用镍金属螯合(Ni-NTA)层析柱进行纯化,透析复性。CCK-8法检测SA-hIL2融合蛋白对PHA刺激的人外周血淋巴细胞的增值活性,流式细胞仪分析SA-hIL2融合蛋白对生物素化的B16.F10肿瘤细胞表面锚定修饰效率。结果:SA-hIL2在大肠杆菌中实现了高效表达,约占菌体总蛋白的20%,制备的SA-hIL2融合蛋白纯度达到95%,并具有双重活性,即hIL-2促进PHA刺激的人外周血淋巴细胞的增值活性和SA介导的高效结合至已生物素化的B16.F10肿瘤细胞表面的功能(表面锚定修饰效率约95%)。结论:研制的SA-hIL2融合蛋白具有双重活性,可为研制表面修饰的新型肿瘤细胞疫苗提供基础。

Objective： To prepare and characterize streptavidin-tagged human interleukin-2 （SA-hIL2） fusion protein. Methods： pET24a-6His-SA-hIL2 plasmid was constructed and expressed in BL 21 （DE3） host bacteria to generate fusion protein. The recombinant fusion protein SA-hIL2 was purified through the Ni-NTA affinity chromatography, and then refolded. The efficiency of surface modification of the fusion protein on the biotinylated BI6. F10 tumor cells was evaluated by a flow cytometer. CCK-8 method was used to determine the proliferating effect of SA-hIL2 fusion protein on human peripheral-blood lymphocyte （PBL） cells stimulated by PHA. Results： The recombinant SA-hIL2 fusion protein was highly expressed in BL21 （DE3） at up to 20% of total bacterial proteins. The fusion protein SA-hIL2 exhibited the bi-functionality： proliferation promoting activity of hlL-2 on PBL cells, and SA-mediated high-affinity binding to the biotinylated surface of B16. F10 cells with about 95% surface modification efficiency. Conclusion： SA-IL2 bi-functional fusion protein was generated, which will make feasible the development of hIL2-surtface-modified cancer cell vaccine.