人IL-18突变体D134R的构建及其生物学活性分析  

The Construction of Human IL-18 Mutant D134R and Its Effects on hIL-18 Biological Activity

作　　者：傅奕[1] 裴冬生[2] 陆梁[2] 胡书群[2] 赵惠仁[2] 

机构地区：[1]扬州大学医学院生化教研室,江苏扬州225001 [2]徐州医学院生物化学与分子生物学研究中心,江苏徐州221002

出　　处：《实用临床医药杂志》2008年第3期24-27,41,共5页Journal of Clinical Medicine in Practice

基　　金：江苏省自然科学基金资助项目(00KJB310008)

摘　　要：目的研究IL-18结构与功能的关系。方法用重叠延伸PCR定点突变技术构建人白介素18(hIL-18)突变体hIL-18D134R。将突变体cDNA与原核表达载体pJW2重组并转化大肠杆菌DH5α。经热诱导表达蛋白质,SDS-PAGE证实,表达的目的蛋白质以包涵体形式存在。菌体经超声破碎后,包涵体以2 mol/L尿素洗涤,8 mol/L尿素溶解,并经Sephadex G-75柱纯化及稀释、透析复性。然后以诱导人外周血单个核细胞(PBMC)产生干扰素-γ(IFN-γ)及对核因子-κB(NF-κB)的激活能力为指标,检测突变体的生物学活性。结果构建的突变体hIL-18D134R可在大肠杆菌DH5α中高效表达,经包涵体洗涤和Sephadex G-75柱纯化后,纯度达92%以上。突变体D134R对IFN-γ的诱生能力及对NF-κB的激活能力分别为野生型hIL-18的19%和23%。结论在人IL-18的氨基酸序列中,Asp134对其生物学功能有非常重要的作用。Objective To study the relationship between structure and function of human IL-18（hIL-18）. Methods site-directed mutagenesis was used to construct hIL-18 mutant D134R. The cDNAof D134R was cloned into prokaryotic expression vetor pJW2 and expressed efficiently in inclusion bodies in E. coli DHSa. The inclusion bodies were washed with 2 mmol/L urea, dissolved in 8 mmol/L urea, and then purified by Sephadex G-75 chromatography. The activities of wild type and mutant hIL-18 were defined as the ability to induce interferon-gamma （IFN-γ production from human peripheral blood mononuclear cells （PBMC） and the effect on nuclear factor-κB（NF-κB） acti- vation. Results The constructed hIL-18D134R can be highly expressed in E. coli DHSα. The purity of the recombinant protein after renaturation was over 92 % as judged by SDS-PAGE. Furthermore, our results showed that D134R not only induced significantly less amount of IFN-γ from PBMC （19% of wild type hIL-18）, but also showed much lower level of NF-κBactivation（23% of wild type hIL-18）. Conclusion The Asp134 is critical for the function of human IL-18.

关 键 词：白细胞介素18 定点突变 干扰素-Γ 核因子-ΚB 

分 类 号：R392.11[医药卫生—免疫学]