重组人IL-18和IL-18-GFP融合蛋白的表达及其生物学活性的研究  

Expression and analysis of recombinant human IL 18 and human IL 18 GFP fusion protein

作　　者：胡晓[1,2] 吕平[1,2] 王丽峰 高晓明[1,2] 

机构地区：[1]北京医科大学免疫学系 [2]解放军463医院三内科

出　　处：《中华微生物学和免疫学杂志》1999年第4期306-310,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家教委回国留学人员重点基金

摘　　要：目的在大肠杆菌中表达重组人ＩＬ－１８（ｒｈＩＬ－１８）及ｒｈＩＬ－１８－绿色荧光蛋白（ＧＦＰ）融合蛋白并测定其生物活性。方法构建原核表达质粒ｐＥＴ２８ａ／ｈＩＬ－１８及ｐＴｒｃ９９／ｈＩＬ－１８－ＧＦＰ，并分别转化大肠杆菌ＢＬ２１。用ＩＰＴＧ诱导重组蛋白的表达，超声裂菌上清中的ｒｈＩＬ－１８用镍金属螯合层析柱进行纯化，ｒｈＩＬ－１８－ＧＦＰ用抗ＧＦＰ亲和层析柱及ＳｅｐｈａｃｒｙｌＳ－１００分子筛柱纯化。以人的外周血单核淋巴细胞测定两重组蛋白的生物活性。结果表达的ｒｈＩＬ－１８及ｒｈＩＬ－１８－ＧＦＰ均具有促进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，ｒｈＩＬ－１８－ＧＦＰ还具有ＧＦＰ的绿色荧光特性，可对Ｐ８１５细胞进行标记。结论表达并纯化的ｒｈＩＬ－１８及ｒｈＩＬ－１８－ＧＦＰ具有生物活性，可用于进一步的功能研究。Objective Interleukin 18 (IL 18), also known as IFN γ inducing factor, shares certain functional properties with IL 12. To study the biologic activities and receptor distribution of recombinant human IL 18 and IL 18 GFP fusion protein expressed in E. coli. Methods Two prokaryotic expression plasmids, pET28a/hIL 18 and pTrc99/hIL 18 GFP have been constructed to express the recombinant human IL 18 (rhIL 18) and its fusion protein with green fluorescent protein (GFP) in E. coli BL21, respectively. rhIL 18 was tagged with six histidine residues at its C terminus and was purified by nickel chelation chromatography. rhIL 18 GFP was purified with anti GFP affinity chromatography and gel filtration. Biological activities were assayed with human peripheral blood mononuclear cells (PBMC). Results Preliminary studies showed that rhIL 18 GFP as well as rhIL 18 augmented T cell proliferation and IFN γ production by ConA stimulated human PBMCs, and enhanced NK cell cytotoxicity. In addition, rhIL 18,GFP has shown specific binding to P815 cells and the stained cells could be analyzed with flow cytometry. Conclusions The rhIL 18 and rhIL 18 GFP we prepared are functional and can be applied to further studies on human IL 18.

关 键 词：IL-18 绿色荧光蛋白 融合蛋白 IFN-γ诱导因子 

分 类 号：R392.1[医药卫生—免疫学]