重组人IL-6D24-PE40外毒素融合蛋白的构建及其生物学效应

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－6D24－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24个氨基酸的重组人白细胞介素6（IL－6D24）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－6D24－PE40融合基因。利用HB101／pBV220表达系统，实现了IL－6D24－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－6D24－PE40融合蛋白在大肠杆菌中的表达水平达到40％－60％。包涵体蛋白经分离、复性及纯化得到纯度＞95％的融合蛋白。Western blot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－6D24－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250ng/ml,而对不表达IL－6R的CEM细胞无杀伤作用。结论 IL－6D24－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。

Construction of recombinant human interleukin-6-Pseudomonas exotoxin fusion proteins and study on their biological characters

Objective To construct a human recombinant interleukin 6 (IL 6) Pseudomonas exotoxin IL 6D24 PE40 fusion protein and to analyze its ability to selectively kill cancer cells and leukemia cells expressing high levels of IL 6 receptors. Methods cDNA encoding the human IL 6 devoid of N terminal 24 amino acids (IL 6D24) was ligated with DNA fragment coding the Pseudomonas exotoxin devoid of its cell recognition domain (PE40). The resultant genes were ligated into expression vector pBV220 and then transformed into E.coli HB101 cells. The expressed recombinant protein was purified to electrophoresis purity by Mono Q column. Its cytotoxicity was measured by the MTS colorimetric method using U937 cells as targets. Results Fusion protein IL 6D24 PE40 was expressed in inclusion bodies experiments at levels of 40% 60% of total proteins in bacterial cells. Western blotting showed that the purified product could react with IL 6 antibody and PEA antibody, respectively. IL 6D24 PE40 were specifically cytotoxic to U937 cells with ID 50 of 250ng/ml and non cytotoxic to IL 6 receptor negative cell line CEM. Conclusion IL 6D24 PE40 fusion protein and derivatives can selectively kill the target cells which express high levels of IL 6 receptors.