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

Keratinocyte growth factor 2 is a member of fibroblast growth factor family found recently, also known as FGF10 .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,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 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 23kDa. The recombinant protein was purified by Ni+-NTA column ,and the purity of 6×his- hKGF2 protein reached over 95% after affinity chromatography . Modified tertrozalium salt(MTT) assay showed that the recombinant protein could signigicantly promote proliferation of human embryonic kidney epithelial cells .