重组人KGF-1蛋白的表达、纯化及活性研究

摘要

The aim of this study was to express soluble recombinant human keratinocyte growth factor -1(rhKGF-1) in Escheri chia coli and to determine the bioactivity of rhKGF-1 to provide the basis for developing new drugs containing rhKGF-1.The sequence encodingΔN23 rhKGF-1(the form of KGF-1 missing 23amino acids in N terminal) was synthesized and fused with SUMO through overlap PCR to obtain 6His-sumo-rhKGF-1 fusion gene subcloned into expression plasmid pET 30a.The recombinant plasmid pET30a-6His-sumo-rh-KGF-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 rhKGF1.TheΔN23 rhKGF1 was used to pro-mote proliferation of HaCat cells , then to assay its bioactivity by CCK-8 methods.As results, the recombiant expression plasmid pET30a-6His-sumo-rhKGF1 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 purifiedΔN23 rhKGF-1 had been obtained.It could promote HaCat cells proliferation , and the value of EC 50 was 6.11 ng/mL.%在大肠杆菌中表达可溶性的重组人rhKGF-1蛋白，研究其生物学活性，为rhKGF-1蛋白进一步制药提供基础。合成ΔN23 rhKGF-1（N端缺失23个氨基酸残基的rhKGF-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pET30a-6His-sumo-rhKGF1，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 rhKGF-1蛋白。 CCK-8法测定其对人永生化表皮细胞（HaCat细胞）的促增殖作用。结果表明，成功构建了pET30a-6His-sumo-rhKGF1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 rhKGF-1蛋白。制备的ΔN23 rhKGF-1蛋白具有促进HaCat细胞增殖的作用，EC50＝6.11 ng／mL。