重组hKGF2原核表达载体的构建及其蛋白质的纯化  

Construction of Recombinant Prokaryotic Expression Vector of Human KGF2 and Its Purification

作　　者：余德荣[1] 游力[2] 王郡甫[1] 许晓群[1] 赵跃然[2] 高春义[1] 

机构地区：[1]山东省医学科学院基础医学研究所山东省肿瘤免疫与基因工程重点实验室山东省现代医用药物与技术重点实验室,济南250062 [2]山东大学山东省立医院生殖医学研究中心,济南250021

出　　处：《中国生物工程杂志》2007年第2期9-13,共5页China Biotechnology

基　　金：国家自然科学基金资助项目(30371304)

摘　　要：角质细胞生长因子2(keratinocyte growth factor2,KGF2)是新近发现的成纤维细胞生长因子家族中的一员,又称FGF10,是上皮细胞特异性的促有丝分裂剂,具有广泛的应用前景。为构建其高效原核表达体系,用RT-PCR法从培养的人胚胎肺成纤维细胞中提取成熟hKGF2cDNA,进行T-A克隆;经测序后,构建pET-30a(+)-hKGF2重组表达载体,将其转入大肠杆菌BL21(DE3)菌株。IPTG诱导表达后,重组蛋白经SDS-PAGE及Western blot鉴定为高效可溶性表达。镍亲和层析(Ni+-NTA)一步法纯化后获得纯度达95%以上的目的蛋白,生物活性研究表明,重组hKGF2能有效促进人胚胎肾上皮细胞的增殖。Keratinocyte growth factor 2 is a member of fibroblast growth factor family found recently, also known as FGFIO. KGF2 is a specific mitogen of epithelial cells and has a good extensive application prospect. To construct highly effective prokaryofic expressional pET-30a （＋） vector containing hKGF2 gene, The total RNA was extracted from the cultured human embryonic lung fibroblast . The hKGF2 gene absent signal peptide was amplified by using RT-PCR. Then the hKGF2 gene was inserted into pMD18-T vector, which was confirmed by sequence anlysis, and the hKGF2 gene was cloned into pET-30a （＋） expression vector , then inducing it with 1mmol/L IPTG . The recombinant protein was detected by SDS-PAGE and Westem blot analysis. The 6 × his-hKGF2 protein was successfully expressed and soluble , and the target protein amounted to 25 % of total bacteria proteins and the molecular weight is approximately 23kDa. The recombinant protein was purified by Hi ＋ -NTA column , and the purity of 6 × his- hKGF2 protein reached over 95% after affinity chromatography . Modified tertrozalium salt（MTF） assay showed that the recombinant protein could signigicantly promote proliferation of human embryonic kidney epithelial cells .

关 键 词：重组hKGF2 克隆 原核表达 镍亲和层析 

分 类 号：Q789[生物学—分子生物学]