鹅IL-2基因在大肠杆菌中的表达及其可溶性单体的分离  

Expression of Goose Interleukin-2 gene in Escherichia coli and Isolation of Its Soluble Monomer

作　　者：齐静[1] 陈吉刚[1] 王金勇[1] 方杰[1] 吴佳俊[1] 周继勇[1] 

机构地区：[1]浙江大学动物预防医学研究所病毒与免疫研究室

出　　处：《生物工程学报》2008年第2期183-187,共5页Chinese Journal of Biotechnology

基　　金：国家杰出青年科学基金资助(No．30625030)~~

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)进作逐级分离,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。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 rgolL-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 AKTA 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.

关 键 词：鹅IL-2 原核表达 纯化 单体 

分 类 号：Q78[生物学—分子生物学] S852.4[农业科学—基础兽医学]