人白细胞介素18(hIL-18)的纯化及生物学活性的鉴定  

Purification and biological identification for the recombinant human interleukin 18

作　　者：杨广民[1] 方艳秋[2] 谭岩[2] 刘力华[2] 

机构地区：[1]吉林省人民医院中心实验室,长春130021 [2]吉林大学第一医院中心实验室,长春130021

出　　处：《中国免疫学杂志》2004年第12期850-854,共5页Chinese Journal of Immunology

基　　金：卫生部临床学科重点科研项目 (2 0 0 13 14 5 ) ;吉林省科技厅重点科研项目 (2 0 0 2 0 40 3 ) ;长春市科委科研项目 (0 4 0 2GG2 18) ;2 0 0 1年吉林大学医疗新技术新疗法;2 0 0 2年吉林大学创新基金

摘　　要：目的 :在大肠杆菌中高效表达重组人IL 18(rhIL 18)蛋白 ,制备具有高纯度、高活性的rhIL 18。方法 :用IPTG诱导重组蛋白表达载体pKK2 2 3 3 hIL 18进行蛋白表达 ;菌体经超声破碎分离包含体 ,并对包含体进行洗涤、变性和复性处理 ,用DEAE SepharoseCL 6B阴离子交换柱纯化复性的rhIL 18;以人外周血单核淋巴细胞 ,通过T细胞增殖实验、1 2 5I UdR标记的细胞毒实验、ELISA方法检测细胞因子的产生量等方法 ,测定rhIL 18的生物学活性。结果 :在大肠杆菌中成功地诱导、表达了rhIL 18,SDS PAGE凝胶电泳分离显示在分子量大约 18 3kD处有一诱导蛋白带 ,Westernblot证明表达产物与抗IL 18单克隆抗体有特异性免疫反应 ;表达产物经纯化后纯度达 94 % ;表达的rhIL 18蛋白具有促进T细胞增殖、增强NK细胞细胞毒作用及诱导外周血单核淋巴细胞合成IFN γ的能力 ,基本上具有天然IL 18相同的生物学活性。结论 :为rhIL 18的获得及为进一步研究其生物学功能提供了一定的条件 ,并为将rhIL 18用于肿瘤的免疫生物治疗奠定了基础。Objective:To express recombinant human IL 18 in E.coli strain and to purify the target cytokine with high purity and bioactivity.Methods:The encoding sequence for the mature peptide of human IL 18 (rhIL 18) was cloned to establish a prokeryotic expressing plasmid with pKK233 3 as the vector. The expression of rhIL 18 was induced by IPTG incubation of the recombinant host strains. The induced product was identified using SDS PAGE and Western blot and purified from inclusion body by DEAE Sepharose gel filtration after the renaturing treatment. Biological activities was analysed with human PBMNCs.Results:Human IL 18 is expressed after IPTG induction, which was indicated as a 18 3 kD band recognized by anti human IL 18 moAb in SDS PAGE and western blot. The purity reaches up to 94% after column chromatogramph. Preparations of the rhIL 18 stimulates proliferation of T cells, induces secretion of IFNγ by the monocytes and lymphocytes in peripheral blood and enhances NK cytotoxicity of the PBMNCs.Conclusion:Highly purified rhIL 18 is obtained by recombinant prokeryotic expressing system,which will become the basis for the immunotherapeutic regimen to cancer. [

关 键 词：重组人白细胞介素18 凝胶过滤 T细胞 NK细胞 细胞因子 

分 类 号：R392.1[医药卫生—免疫学] R73-35[医药卫生—基础医学]