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…

生物工程学报 Chin J Biotech 2008, February 25; 24(2): 183-187

journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061

Received: April 24, 2007; Accepted: June 14, 2007

Supported by: the National Natural Science Funds for Distinguished Young Scholar (No.30625030).

Corresponding author: Jiyong Zhou. Tel: +86-571-86971698; E-mail: jyzhou@zju.edu.cn

国家杰出青年科学基金资助(No.30625030)。

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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