摘要目的 通过构建肝细胞生长因子(HGF)基因的重组原核表达载体,转化大肠杆菌,制备HGF重组蛋白并初步证实其活性.方法 克隆HGF基因插入载体pET-26b(+),构建重组原核表达载体pET-26b(+)-HGF,转化E.coli Rosseta(DE3).IPTG诱导转化菌表达重组蛋白,采用Ni-NTA树脂亲和层析法进行纯化,复性后冷冻干燥.结果 HGF基因重组原核表达载体pET-26b(+)-HGF构建成功.转化pET-26b(+)-HGF的E.coli Rosseta(DE3)以包涵体形式大量表达目的蛋白,占菌体总蛋白的38％,并经Western blot证实.经Ni-NTA树脂亲和层析法纯化后HGF蛋白纯度约为95％,作用于人非小细胞肺癌细胞系A549细胞发现可促进其增殖、迁徙并抑制凋亡.结论 成功构建了HGF基因重组原核表达载体pET-26b(+)-HGF,转化E.coli Rosseta(DE3)后成功表达,纯化复性回复重组HGF蛋白结构,经体外实验证实具有生物学活性.

abstractsObjective To prepare hepatocyte growth factor(HGF) recombinant protein and confirm its activity preliminarily according to building HGF gene prokaryotic expression vector and transforming into E.coli.Methods Clone HGF inserted into the vector pET-26b(+) to construct prokaryotic expression vector pET-26b(+)-HGF and transform into E.coli Rosseta(DE3).The transformed bacteria induced by IPTG was purified through Ni-NTA resin affinity chromatography frozen-drying after renaturation.Results HGF gene recombinant prokaryotic expression vector pET-26b(+)-HGF was constructed successfully.E.coli Rosseta(DE3) which was transformed into pET-26b(+)-HGF expresses the target protein as the form of inclusion bodies,accounting for 38％ of the total bacterial proteins,and confirmed by Western blot.HGF protein which was purified by Ni-NTA resin affinity chromatography,has a purity of about 95％,and can promote proliferation,migration,and inhibition of apoptosis for human non-small cell lung cancer cell line A549 cells after interaction.Conclusion HGF gene recombinant prokaryotic expression vector pET-26b (+)-HGF was constructed and expressed in transformed E.coli Rosseta(DE3) successfully.They resumed their recombinant HGF protein structure after purification and renaturation,and had biological activity confirmed by in vitro studies.