【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。更多还原

【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 sys- tem,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 purifi- cation 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.更多还原