链霉亲和素-人粒细胞巨噬细胞集落刺激因子融合蛋白的制备与活性鉴定

Preparation and bioactivity evaluation of SA-hGM-CSF fusion protein

目的:制备链霉亲和素(SA)连接的人粒细胞巨噬细胞集落刺激因子(hGM-CSF)融合蛋白(SA-hGM-CSF),并对其生物学活性进行鉴定。方法:在NCBI数据库中查找SA和hGM-CSF序列,将序列合成后连接入Pet28表达载体中,构建SA-hGM-CSF-Pet28重组表达载体,在BL21(DE3)表达感受态中利用IPTG诱导剂诱导表达,经His镍柱纯化,尿素梯度复性,聚丙烯酰胺凝胶电泳法和蛋白质印迹法(WB)鉴定融合蛋白,通过TF-1细胞检测融合蛋白的生物学活性。结果:SA-hGM-CSF融合蛋白可在BL21(DE3)中高效诱导表达,经His镍柱纯化后该融合蛋白纯度较高。经尿素梯度复性后,细胞学实验验证该融合蛋白具有生物学活性。结论:成功表达具有生物活性的融合蛋白SA-hGM-CSF,该结果为SA-hGM-CSF表面锚定修饰的肿瘤细胞疫苗的研究提供了实验基础。

Objective:To prepare the streptavidin-tag hGM-CSF fusion protein SA-hGM-CSF,and to identify its biological activity.Methods:The sequences of SA and hGM-CSF were found in NCBI,and the sequences were synthesized and connected to Pet28 expression vector to construct the recombinant expression vector of SA-hGM-CSF-Pet28,which was induced by IPTG inducer in BL21(DE3)expression induced state,purified by His nickel column.After renaturation by urea gradient,the fusion proteins were identified by SDS-PAGE and Western blot.The biological activity of the fusion proteins was detected by TF-1 cells.Results:The fusion protein of SA-hGM-CSF could be efficiently induced in BL21(DE3),and the purity of the fusion protein was high after His nickel column purification.After urea gradient renaturation,cytological experiments verified the biological activity of the fusion protein.Conclusion:The fusion protein SA-hGM-CSF was successfully expressed,which provides experimental basis for the study of tumor cell vaccine modified by surface anchoring of SA-hGM-CSF.