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目的 构建重组人白细胞介素 6 绿脓杆菌外毒素融合蛋白IL 6D2 4 PE40 ,以选择性杀伤高表达IL 6受体 (IL 6R)的肿瘤细胞和白血病细胞。方法 采用重叠延伸的基因融合技术将N 末端缺失 2 4个氨基酸的重组人白细胞介素 6 (IL 6D2 4)cDNA与缺失细胞结合区的绿脓杆菌外毒素PE40基因进行融合 ,构建IL 6D2 4 PE40融合基因。利用HB10 1/pBV2 2 0表达系统 ,实现了IL 6D2 4 PE40融合蛋白在大肠杆菌中的高效表达 ,经MonoQ柱进行层析纯化 ,采用MTS法检测细胞毒活性。结果 IL 6D2 4 PE40融合蛋白在大肠杆菌中的表达水平达到 40 %～ 6 0 %。包涵体蛋白经分离、复性及纯化得到纯度 >95 %的融合蛋白。Westernblot证明 ,纯化的融合蛋白与IL 6抗体及PEA抗体均发生特异性结合。细胞毒活性检测证实 ,IL 6D2 4 PE40融合蛋白能高度特异地选择性杀伤高表达IL 6R的U937细胞 ,ID50 约为 2 5 0ng/ml,而对不表达IL 6R的CEM细胞无杀伤作用。结论 IL 6D2 4 PE40融合蛋白能够选择性杀伤高表达IL 6R的靶细胞 ,有希望成为导向治疗高表达IL 6 /IL 6R系统的某些白血病和其他肿瘤的新型导向药物
Objective To construct recombinant human interleukin - 6 (Pseudomonas aeruginosa) exotoxin fusion protein IL 6D2 4 PE40 to selectively kill tumor cells and leukemia cells that express IL 6 receptor (IL 6R). Methods Recombinant human interleukin 6 (IL 6D2 4) cDNA with N-terminal deletion of 24 amino acids was fused with PE48 gene of Pseudomonas exotoxin in cell-free zone by overlap-extension gene fusion to construct IL 6D2 4 PE40 Fusion gene. The expression of IL-6D2 4 PE40 fusion protein was efficiently expressed in E. coli using the HB10 1 / pBV220 expression system. The purified protein was purified by chromatography on a MonoQ column and the cytotoxic activity was detected by MTS assay. Results The expression level of IL 6D2 4 PE40 fusion protein in E. coli reached 40% ~ 60%. Inclusion body protein was isolated, renatured and purified to obtain the purity of> 95% of the fusion protein. Westernblot demonstrated that the purified fusion protein specifically bound both the IL6 antibody and the PEA antibody. Cytotoxic activity assay confirmed that the IL 6D2 4 PE40 fusion protein could highly selectively selectively kill U937 cells with high expression of IL-6R with an ID50 of about 250 ng / ml, but no cytotoxic effect on CEM cells that do not express IL-6R. Conclusion The IL 6D2 4 PE40 fusion protein can selectively kill target cells with high expression of IL-6R and is promising as a new drug for targeting some leukemias and other tumors that are highly expressed in the IL 6 / IL 6R system