生物工程学报
journals.im.ac.cn cjb@im.ac.cn
Chin J Biotech 2008, February 25; 24(2): 183-187 Chinese Journal of Biotechnology ISSN 1000-3061 2008 Institute of Microbiology, CAS & CSM, All rights reserved
研究报告
鹅 IL-2 基因在大肠杆菌中的表达及其可溶性单体的分离
齐 静 1,2, 陈吉刚 1, 王金勇 1, 方 杰 1, 吴佳俊 1, 周继勇 1
1 浙江大学动物预防医学研究所病毒与免疫研究室, 杭州 310029 2 山东省农业科学院畜牧兽医研究所, 济南 250100
摘
要: 将 去 除 信 号 肽编 码 序列 的 鹅 IL-2 基 因 克 隆 到 原 核 表 达 载 体 pET-28a (+), 构 建 了 重 组 表 达 质 粒 pET-28a
(+)-goIL-2, 转化大肠杆菌 BL21(DE3)感受态细胞, 经 IPTG 诱导, 实现了重组鹅 IL-2(rgoIL-2)蛋白在大肠杆菌中的表达. SDS-PAGE 和 Western-blotting 分析显示, 表达蛋白的分子量约为 15.0 kD, 能被抗鹅 IL-2 单克隆抗体特异识别.可溶性 分析表明表达蛋白大部分以包涵体形式存在, 部分以可溶形式存在, 非变性电泳可见可溶性蛋白存在单体和多聚体组 分.镍柱亲和层析法纯化的 rgoIL-2 蛋白过滤后, 利用 KTA FPLC(快速蛋白分离纯化系统)进行逐级分离, 非变性电泳 可见单一的鹅 IL-2 可溶性蛋白单体.体外生物学活性分析显示鹅 IL-2 可溶性蛋白单体能刺激鹅淋巴细胞增殖.这为进 一步研究鹅 IL-2 的生物学功能及其临床应用奠定基础. 关 键 词 : 鹅 IL-2, 原 核 表 达 , 纯 化 , 单 体
Expression of Goose Interleukin-2 gene in Escherichia coli and Isolation of Its Soluble Monomer
Jing Qi1,2, Jigang Chen1, Jinyong Wang1, Jie Fang1, Jiajun Wu1, and Jiyong Zhou1
1 Laboratory of Virology and Immunology, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029, China 2 Institute of Animal and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China
Abstract: Recombinant expression plasmid of pET-28a (+)-goIL-2 was constructed by inserting the goose IL-2 gene without the
signal peptide sequence into the prokaryotic expression vector pET-28a (+), and transformed into the bacterial competent E. coli BL21 (DE3) cells for expression. After IPTG induction, an expected protein band with molecular weight of 15.0 kD was observed on SDS-PAGE gel, recognized by monoclonal antibody against goose IL-2 in western-blotting assay. In the pET-28a (+) expression system, much of the recombinant goose IL-2 (rgoIL-2) was found in inclusion bodies with a portion of soluble protein. The monomer and multimers of soluble goose interleukin 2 proteins were observed in native electrophoresis. The rgoIL-2 proteins were purified by Ni-NTA column under a native condition. The rgoIL-2 soluble protein monomer was isolated by a quick protein isolation and purification system of KTA FPLC and identified by native PAGE. Bioactivity analysis showed that the rgoIL-2 monomer stimulated the proliferation of goose lymphocytes in vitro. This will establish a basis for further study about the biological function and clinical application of goose IL-2. Keywords: goose interleukin 2, prokaryotic expression, purification, monomer

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