一种具杀菌功能的haFGF融合基因在毕氏酵母中的表达

目的构建一种双功能活性多肽,实现其在酵母中的表达。方法通过体外DNA重组技术将cecropin AD连接在haFGF的5′端,构建成融合基因CADAF,将其克隆到分泌表达载体pPICZαA上,然后转化到毕氏酵母受体菌X-33中进行甲醇诱导表达。结果经SDS PAGE和Western blot分析,构建的CADAF基因在酵母中获得了表达,经抑菌实验及MTT实验结果表明,融合蛋白具有一定的抗菌功能和促细胞分裂活性。结论成功实现了haFGF与抗菌肽的在酵母中的融合表达,为多功能细胞生长因子在创伤治疗中的应用打下了基础。

The Fusion Expression of a Antimicrobial Peptide haFGF in Pichia Pastoris Cell

Objective Constructed a dual function peptide,and made it expressed in yeast.Methods Cecropin AD was connected with 5′end of the cDNA of human acidic Fibroblast Growth Factor（haFGF） by using DNA recombination in vitro,and then CADAF fusion gene was cloned into pPICZαA resulting in a secretion expression vector.The recombinant plasmid was transformed into Pichia pastoris X-33,which was induced by methanol induction.Results SDS-PAGE and Western blot analysis showed the fusion gene CADAF was expressed in Pichia pastoris successfully,It was confirmed that CADAF protein has the immunogenicity of haFGF.The analysis of restraining the growth of E·coliK12D31 showed CADAF has the antimicrobial activity.MTT results indicated CADAF can stimulate Balb/c3T3 cells proliferation.Conclusion aFGF-Cecropin fusion peptide have been expressed in yeast successfully.This study provides a foundation for the use of multifunctionin factor in wound cured.