摘要
目的制备链亲和素(SA)/人白细胞介素-15(hIL-15)融合蛋白SA-hIL15和hIL-15-SA,并鉴定其生物学活性。方法构建原核表达质粒pET24a-6His-SA-hIL-15和pET32a-hIL-15-SA-6His,转化大肠杆菌BL21(DE3),用镍金属螯合(Ni-NTA)层析柱进行纯化,并对其进行复性。CCK-8检测融合蛋白PHA刺激的人外周血淋巴细胞增殖的活性,流式细胞仪分析融合蛋白对生物素化的B16.F10细胞锚定修饰率。结果成功构建了两种重组融合蛋白的表达质粒并在大肠杆菌实现了高效表达,目标蛋白的表达约占细菌总蛋白的20%。经纯化、复性的SA/hIL-15融合蛋白具有双重活性,即:能促进PHA激活的人外周血淋巴细胞增殖的hIL-15活性,和SA介导的高效结合至表面已生物素化的B16.F10肿瘤细胞的功能(表面锚定修饰效率大于95%)。SA-hIL-15双功能融合蛋白促进PHA激活的人外周血淋巴细胞增殖活性高于hIL-15-SA双功能融合蛋白。结论SA/hIL-15双功能融合蛋白的制备为hIL-15表面锚定修饰的肿瘤细胞疫苗的研制打下了基础。
Objective To obtain streptavidin-tagged human interleukin-15 （SA/hIL15） fusion protein and evaluate its bioactivity. Methods pET24a-6His-SA-hIL-15 and pET32a-hIL-15-SA-6His plasmids were constructed and expressed in BL 21 （DE3） host bacteria to generate the fusion protein. The recombinant fusion protein IL-15/SA was purified using Ni-NTA affinity chromatography and refolded, and the efficiency of surface modification of the fusion protein on biotinylated cells was examined by fluorescence-activated cell sorting. CCK-8 method was used to evaluate the effect of IL-15/SA fusion protein in inducing the proliferation of human peripheral-blood lymphocyte （PBL） cells stimulated by PHA. Results The recombinant SA-hIL-15 and hIL15-SA fusion proteins were highly expressed in BL21 （DE3） at about 20% of the total bacterial proteins. The purified hiLl 5-SA fusion protein exhibited a bifimctionality by promoting the proliferation of PBL cells activated by PHA and high-affinity binding to biotinylated cell surface mediated by SA, with a cell surface modification efficiency exceeding 95%. SA-hIL-15 showed a 4-fold higher hiLl5 bioactivitythan hIL15-SA. Conclusion SA/hIL-15 bifunctional fusion protein has been successfully obtained to facilitate the future development of hIL-15-surtface-modified cancer cell vaccine.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2009年第3期397-401,共5页
Journal of Southern Medical University
基金
国家“863”计划(2006AA02Z4C4)
广东省自然科学基金(06301184)
