【摘要】 为研究IL 18结构与功能的关系 ,用重叠延伸PCR定点突变技术构建人白细胞介素 18(hIL 18) 4个半胱氨酸的突变体hIL 18C74 S、C10 4 S、C112 S和C163 S。将突变体的cDNA与原核细胞表达载体pJW2重组并转化大肠杆菌JM10 1。经热诱导后 ,4个突变体在大肠杆菌中均得到了高效表达。表达的蛋白质主要以包涵体的形式存在。包涵体经超声破碎 ,2mol/L尿素洗涤 ,8mol/L尿素溶解 ,SephadexG 10 0柱纯化后 ,纯度可达 90 %以上。以诱导人外周血单个核细胞 (PBMC)产生IFN γ的能力为指标检测复性突变体的活性。结果显示除C10 4 S外 ,其他 3个突变体的生物活性均低于野生型hIL 18,C74 S、C112 S和C163 S的活性分别是野生型hIL 18活性的 5 %、5 8%和11%。证明Cys74 、Cys163 为hIL 18诱导产生IFN γ的功能所必需更多还原

【Abstract】 To study the structure-function relationships of human IL-18(hIL-18), site-directed mutagenesis was used to generate four hIL-18 cysteine mutants, C 74S, C 104S, C 112S and C 163S. The cDNAs of the four cysteine mutants were inserted into prokaryotic expression vector pJW2 and expressed as inclusion bodies in E.coli. The inclusion bodies were washed with 2 mol/L urea, dissolved in 8 mol/L urea, and purified by chromatography on Sephadex G-100 column. The purity of the purified mutants were greater than 90% as judged by SDS-PAGE. The activity of rhIL-18 C 74S, C 104S, C 112S and C 163S accounted for 5%, 81%, 58% and 11% of wild type, respectively. These results suggest that Cys 74 and Cys 163 play important roles in inducing IFN-γ production in human peripheral blood mononuclear cells.更多还原