人白细胞介素18突变体的构建及其功能(英文)  

Identification of Important Amino Acid Residues for Human IL-18 Function by Mutant Construction

作　　者：傅奕[1] 裴冬生[2] 孙兵[3] 沈万华[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,扬州225001 [2]徐州医学院生物化学与分子生物学研究中心,徐州221002 [3]苏州大学附属医院神经外科,苏州215001

出　　处：《生物化学与生物物理学报》2003年第5期409-415,共7页

摘　　要：为研究人白细胞介素 18(hIL 18)结构与功能的关系 ,用PCR定点突变技术分别构建了N末端、C末端缺失突变体(ΔNC)和IL 1特征样序列突变体S154A/Y156F/E157P/C163 T (S)。将突变体cDNA与原核表达载体 pJW 2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 2mol/L尿素洗涤 ,8mol/L尿素溶解 ,并经SephadexG 75柱纯化 ,纯度可达 95 %以上。突变体蛋白质经逐步稀释复性后 ,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。结果显示ΔNC、S这 2个突变体对IFN γ的诱生能力显著低于野生型hIL 18,分别为野生型hIL 18的 13%和 4 8%。同时 ,ΔNC、S对NF κB的激活能力也低于野生型hIL 18,分别为野生型hIL 18的 6 9.7%和 89.8%。这些结果表明缺失的片段或突变的位点对hIL 18的功能有重要的作用。To study the structure-function relationship of IL-18, two IL-18 mutants, N- and C-terminal mutant (ΔNC) and IL-1 signature-like sequence mutant S 154A/Y 156F/E 157P/C 163T (S), were constructed by PCR. The wild type and mutant recombinant human interleukin-18 (rhIL-18) were expressed in E.coli, purified by Sephadex G-75 chromatography and renatured by stepwise dilution. The purity of the recombinant proteins was over 95%. The activities of wild type and mutant rhIL-18s were defined as the ability to induce interferon-gamma (IFN-γ) production and NF-κB activation from human peripheral blood mononuclear cells (PBMC). Our results showed that the two mutants induced significantly less amount of IFN-γ from PBMC (13%, 48% of wild type rhIL-18 for ΔNC , S respectively), and the activation of NF-κB also lower than wild type rhIL-18(69.7%, 89.8% of wild type rhIL-18 respectively), indicating that the deleted or mutated amino acids might be important for IL-18 function.

关 键 词：人白细胞介素18 hIL-18 突变体 干扰素 定点突变 基因结构 

分 类 号：Q78[生物学—分子生物学]