SA/hIL21双功能融合蛋白的表达、纯化及生物学活性检测

目的 制备链亲和素(SA)标记的人白细胞介素21融合蛋白(SA-hIL-21),检测其生物学活性.方法 构建hIL21-SA-pET21及pET24a-SA-hIL21质粒,利用大肠杆菌BL21(DE3)表达两种双功能融合蛋白,并利用镍金属螯合层析柱(Ni-NTA)纯化,之后透析复性,Western blotting进行鉴定,最后利用MTT法检测hIL21-SA及SA-hIL21融合蛋白与抗CD3单克隆抗体(anti-CD3)共刺激人外周血淋巴细胞的增殖活性,流式细胞仪分析两种融合蛋白对生物素化的MB49肿瘤细胞的锚定修饰效率.结果 hIL21-SA及SA-hIL21重组融合蛋白在大肠杆菌中实现了高效表达,约占菌体蛋白的30%,经复性后hIL21-SA及SA-hlL21具有了双重活性,即不仅可以与抗CD3单抗共刺激淋巴细胞的增殖,而且具有了SA介导的高效结合已生物素化的MB49肿瘤细胞表面的功能(表面修饰效率95.18%,96.91%).结论 本实验成功研制了具有双重活性的hIL21-SA及SA-hlL21融合蛋白,两种融合蛋白的双功能活性无显著性差异,该项研究为SA/hIL21双功能融合蛋白应用于肿瘤疫苗以及肿瘤局部治疗奠定了基础.

Expression, purification and bioactivity evaluation of streptavidin-tagged human interleukin-21 fusion protein

Objective To obtain streptavidin-tagged human interleukin-21(hIL21) fusion protein and evaluate its bioactivities.Methods hIL21-SA-pET21 and pET24a-SA-hIL21 plasmids were constructed and expressed in BL21(DE3) host bacteria.The hIL21-SA and SA-hIL21 fusion protein were purified through Ni-NTA affinity chromatography and refolded by dialysis.Flow cytometry was used to detect hIL21-SA and SA-hIL21 fusion protein on the biotinylated MB49 tumor cells.MTT assay was used to evaluate the effect of the fusion prote...