重组截短型人角质细胞生长因子-1的表达及纯化

目的：构建高效表达N端缺失的23个氨基酸重组人角质细胞生长因子1( rhKGF1_dest23)的基因工程菌，为治疗放化疗后口腔黏膜炎的新药开发提供实验数据。方法：利用PCR方法分别以pET3c-hKGF1及sumo-EGF为模板合成N端缺失的23个氨基酸rhKGF1_dest23及sumo基因片段，构建4种原核表达载体pET22b-rhKGF1_dest23﹑pET22b-sumo-rhKGF1_dest23﹑pET3c-rhKGF1_dest23和pET3c-sumo-rhKGF1_dest23，分别转化原核表达宿主菌Rosetta(DE3) plysS、BL21(DE3) 、BL21(DE3)Star plysS、origima(DE3) 和BL21AI，筛选rhKGF1_dest23蛋白表达的最佳质粒与宿主菌组合。CM离子交换和肝素亲和层析法纯化rhKGF1_dest23蛋白，Western blotting法鉴定rhKGF1_dest23蛋白。结果： pET22b-rhKGF1_dest23质粒与BL21AI 宿主菌为最佳组合表达，经IPTG与阿拉伯糖诱导， SDS-PAGE电泳后表明目的蛋白大部分以可溶性形式存在，约占菌体总蛋白的10％，经CM和肝素两步纯化后rhKGF1_dest23蛋白纯度为95%以上，Western blotting法鉴定为rhKGF1_dest23蛋白。结论：成功构建表达人KGF1_dest23重组蛋白的基因工程菌，经IPTG与阿拉伯糖诱导，CM弱阳离子与肝素亲合层析后，得到了纯化的rhKGF1_dest23蛋白。

Expression and purification of recombinant truncated human keratinocyte growth factor-1

Objective To construct the genetic engineering bacteria highly expressing 23 amino acids human keratinocyte growth factor-1(rhKGF1dest23)missing N terminal,and provide experimental data for development of new drug for treatment of oral mucositis after radiotherapy and chemotherapy.Methods PCR was used to synthese 23 amino acids rhKGF1dest23 missing N terminal and sumo gene fragments,and construct four kinds of recombinant prokaryotic expression vectors:pET22b-rhKGF1dest23,pET22b-sumo-rhKGF1dest23,pET3c-rhKGF1dest23 and pET3c-sumo-rhKGF1dest23,then they were transformed into prokaryotic expression host bacteria:Rosetta(DE3)plysS,BL21(DE3),BL21(DE3)Star plysS,origima(DE3)and BL21AI,the best expression combination of plasmid and host strain of rhKGF1dest23 protein was screened and purified by CM ion-exchange and heparin affinity chromatography and identified with Western blotting.Results pET22b-rhKGF1dest23 plasmid and the BL21AI host bacteria was the best combination of expression,after induced by IPTG and arabinose,the majority of recombinant protein was expressed in soluble form,accounting for about 12% of the total bacterial proteins.Its purity reached to more than 95% of the protein after two steps chromatography,then conformed with Western blotting.Conclusion Human genetic engineering bacteria of KGF1dest23 is successfully constructed and induced by IPTG and arabinose,then after CM weak cation exchange and heparin affinity chromatography,the purified rhKGF1dest23 protein is obtained