Abstract:

Objective To construct the genetic engineering bacteria highly expressing  23 amino acids human keratinocyte growth factor -1 (rhKGF1_dest23) 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 rhKGF1_dest23 missing N terminal and sumo gene fragments,and construct four kinds of recombinant prokaryotic expression vectors:  pET22b-rhKGF1_dest23,pET22b-sumo-rhKGF1_dest23,pET3c-rhKGF1_dest23 and pET3c-sumo-rhKGF1_dest23,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 rhKGF1_dest23 protein was screened and purified by CM ion-exchange and heparin affinity chromatography and identified with Western blotting.Results pET22b-rhKGF1_dest23 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 KGF1_dest23 is successfully constructed and induced by IPTG and arabinose,then after CM weak cation exchange and heparin affinity chromatography,the purified rhKGF1_dest23 protein is obtained

Key words: high expression;recombinant truncated human keratinocyte growth factor -1;purification