重组人KGF-1蛋白的表达、纯化及活性研究

Expression,purification and bioactivity detection of recombinant human keratinocyte growth factor-1

在大肠杆菌中表达可溶性的重组人rh KGF-1蛋白,研究其生物学活性,为rh KGF-1蛋白进一步制药提供基础。合成ΔN23 rh KGF-1(N端缺失23个氨基酸残基的rh KGF-1形式)的基因序列,应用重叠PCR技术,将其与poly His-sumo标签基因序列拼接。构建重组表达质粒p ET30a-6His-sumo-rh KGF1,转入E.coli BL21(DE3)中,表达融合蛋白。融合蛋白经Ni-NTA纯化后,切除SUMO标签,再经Ni-NTA纯化获得ΔN23 rh KGF-1蛋白。CCK-8法测定其对人永生化表皮细胞(Ha Cat细胞)的促增殖作用。结果表明,成功构建了p ET30a-6His-sumo-rh KGF1表达质粒,测序结果与预期一致。实现了融合蛋白在E.coli BL21(DE3)中的可溶性表达,经纯化、酶切获得了ΔN23 rh KGF-1蛋白。制备的ΔN23 rh KGF-1蛋白具有促进Ha Cat细胞增殖的作用,EC50=6.11 ng/m L。

The aim of this study was to express soluble recombinant human keratinocyte growth factor-1（ rh KGF-1） in Escherichia coli and to determine the bioactivity of rh KGF-1 to provide the basis for developing new drugs containing rh KGF-1. The sequence encodingΔN23 rh KGF-1（ the form of KGF-1 missing 23 amino acids in N terminal） was synthesized and fused with SUMO through overlap PCR to obtain 6His-sumo-rh KGF-1 fusion gene subcloned into expression plasmid p ET30 a. The recombinant plasmid p ET30a-6His-sumo-rhKGF-1 was transfected to E. coli BL21（ DE3） for expressing the fusion protein. Firstly,the fusion protein was purified by Ni-NTA,then,digested by Sumo Protease,and purified by Ni-NTA again to obtain purified ΔN23 rh KGF1. The ΔN23 rh KGF1 was used to promote proliferation of Ha Cat cells,then to assay its bioactivity by CCK-8 methods. As results,the recombiant expression plasmid p ET30a-6His-sumo-rh KGF1 was constructed correctly,and the result of sequencing was the same as the expected. The fusion protein was expressed in E. coli BL21（ DE3） in soluble form. After purification and SUMO Protease digestion,the purified ΔN23 rh KGF-1 had been obtained. It could promote Ha Cat cells proliferation,and the value of EC50 was 6. 11 ng / m L.