重组hKGF2原核表达载体的构建及其蛋白质的纯化

角质细胞生长因子2（ keratinocyte growth factor 2, KGF2）是新近发现且发展迅速的成纤维细胞生长因子家族中新的一员，又称FGF10，是上皮细胞特异性的促有丝分裂剂，应用前景广泛。为构建其高效原核表达菌株，首先用RT-PCR法从培养的人胚胎肺成纤维细胞中获得去除信号肽的hKGF2 cDNA，将其插入pMD18-T载体；经测序后，克隆入pET-30a( + )表达载体，将成功构建的pET-30a( + )- hKGF2重组表达载体转入大肠杆菌BL21（DE3），经IPTG诱导表达后， SDS-PAGE及Western blot鉴定重组蛋白为高效可溶性表达。重组蛋白经Ni+-NTA（镍亲和层析）一步法纯化后纯度达95%以上，为进一步的应用研究及大规模生产奠定了基础。

Construction of recombinant prokaryotic expression vector of human KGF2 and its purification

Abstract: Keratinocyte growth factor 2 is a member of fibroblast growth factor family, also known as FGF10 ，Which is found recently and developed rapidly .KGF2 is a specific mitogen of epithelial cells and has a good extensive application prospect .To construct highly effective prokaryotic 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 .After DNA sequencing ，the hKGF2 gene was cloned into pET-30a( + ) expression vector ,then induced by IPTG . The recombinant protein was detected by SDS-PAGE and Western 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 23 KD. The recombinant protein was purified by Ni+-NTA column , the purity of 6×his- hKGF2 protein reached over 95% after affinity chromatography .The one affinity column reduces the cost of production and lays a good foundation for large scale production and further exploratory development.