重组人IL-6D24-PE40外毒素融合蛋白的构建及其生物学效应  

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

作　　者：郑黎燕[1] 奚永志[1] 孔繁华[1] 金荔[1] 陈兴国[1] 屠敏[1] 刘楠[1] 

机构地区：[1]军事医学科学院附属医院免疫学研究室,北京100039

出　　处：《中华微生物学和免疫学杂志》2001年第1期95-98,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目! (39870 875 )

摘　　要：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 /ILObjective 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.

关 键 词：重组细胞因子-毒素融合蛋白 IL-6-PE40 基因克隆 生物学效应 

分 类 号：R965[医药卫生—药理学]