γ干扰素诱导蛋白-10的硫氧还基因融合表达、纯化及生物活性检测  

Fusion Expression, Purification and Bioassay of IFN-γ Inducible Protein-10 and Thioredoxin Gene in E.coli

作　　者：李刚[1] 田聆[1] 魏于全[1] 文艳君[1] 肖飞[1] 姚兵[1] 张伶[1] 张汝[1] 梅开[1] 

机构地区：[1]四川大学华西医院人类疾病生物治疗重点实验室.肿瘤中心,成都610041

出　　处：《生物医学工程学杂志》2005年第3期535-539,共5页Journal of Biomedical Engineering

基　　金：国家重点基础研究发展规划(973)前期研究专项资助(2001-50)

摘　　要：γ干扰素诱导蛋白-10是由γ干扰素刺激单核细胞、内皮细胞等所诱导产生趋化T淋巴细胞的一类分泌性糖蛋白。本研究PCR扩增人与鼠γ干扰素诱导蛋白-10的成熟蛋白编码区,克隆到硫氧还载体pET-32(a)进行诱导表达。SDS-PAGE分析显示:在IPTG诱导下,人与鼠的γ干扰素诱导蛋白-10表达量约占目标总蛋白的25.3%,主要以包含体的形式存在,可溶性成分约占目标总蛋白的20%。将可溶性蛋白以S-Tag亲和层析柱进行纯化,纯化的蛋白质纯度约为90%的γ干扰素诱导蛋白-10。通过趋化性分析,证实重组表达的人与鼠γ干扰素诱导蛋白-10在100ng/ml浓度下对激活的T淋巴细胞具有趋化活性。Interferon γ-inducible protein 10, a member of the family of CXC chemokines, is secreted by interferon gamma-stimulated, monocytes, endothelial cells and keratinocytes. Interferon γ-inducible protein 10 plays an important role in recruiting activated T cells into sites of tissue inflammation. In this experiment, PCR products of Interferon γ-inducible protein 10 were cloned into prokaryote expression vector pET 32（a） to generate recombinant pET-IP10 with S-Tag at the N-terminus, and expressed successfully in E. coli BL21 （DE3）, The total expressed products amounted to 25.3% in all bacterion proteins, pET-IP10 mainly formed inclusion body in E. coli. Soluble recombinant protein accounted for 20% among IP-10 fusion protein. The soluble recombinant proteins were purified by using S-Tag affinity chromatography effectively with purity of over 90%. The chemotaxis biological activity of purified Interferon γ-inducible protein 10 could specifically exhibit the directional migration of stimulated T cells at concentration of 100 ng/ml. The results indicated that the strategy we used in this experiment was effective for recombinant Interferon γ-inducible protein 10 production with biological activity.

关 键 词：γ干扰素诱导蛋白-10 硫氧还基因 基因融合 基因表达 基因纯化 生物活性 

分 类 号：Q786[生物学—分子生物学]