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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。
Objective: To prepare SA-labeled murine interleukin-15 fusion proteins SA-mIL15 and mIL15-SA and study their biological functions. Methods: The recombinant plasmids pET24a-SA-L-mIL15 and pET21a-mIL15-L-SA were constructed and expressed in Escherichia coli. The fusion proteins SA-mIL15 and mIL15- -NTA) chromatography and anion exchange (DEAE) chromatography purification, dialysis refolding. The activity of ConA-stimulated splenic lymphocyte proliferation was detected by MTT assay. The efficiency of anchored fusion protein on the surface of biotinylated mouse prostate cancer (RM-1) cells was analyzed by flow cytometry (FCM). Results: SA-mIL15 and mIL15-SA were highly expressed in E. coli, accounting for about 20% of total bacterial proteins. The prepared SA-mIL15 and mIL15-SA fusion proteins have a purity of 95% and have dual activities, that is, mIL15 promotes proliferation activity of ConA-activated mouse spleen lymphocytes and SA-mediated efficient binding to surface biotinylated The function of RM-1 cells (surface anchoring modification efficiency> 95%). Among them, SA-mIL15 bifunctional fusion protein promoted ConA-activated mouse splenic lymphocyte proliferation activity of 1 × 106IU / mg, mIL15-SA activity of 2 × 105IU / mg. Conclusion: The dual activity of SA / mIL15 bifunctional fusion protein lays the foundation for the development of tumor cell vaccine modified by mIL15 surface.