摘要
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-hIL21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-hIL21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。
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 protein on the proliferation of human peripheral blood lymphocytes（PBLs） stimulated by Anti-CD3.Results The recombinant fusion proteins were highly expressed in BL21（DE3） at about 30percnt;of the total bacterial proteins.The two fusion proteins exhibited bifunctional activities,i.e.both biotin-binding property andhIL21 activity and SA-mediated high-affinity binding to biotinylated cell surfaces（with anchoring modified rate of about 95.18percnt;and 96.91percnt;）.Conclusion We have successfully obtained bifunctional fusion protein hIL21-SA and SA-hIL21,which will provide a basis for further study of tumor biotherapy using the proteins.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2010年第6期1240-1243,1249,共5页
Journal of Southern Medical University
基金
国家863高技术研究发展计划(2006AA02Z4C4)
