内生多粘芽孢杆菌EJS-3纤溶酶基因在毕赤氏酵母中的表达

Expression of Paenibacillus polymyxa EJS-3 Fibrinolytic Enzyme Gene in Pichia pastoris

以自构建的重组质粒p ET-Dsb A/PPFE-I为模板,扩增内生多粘芽孢杆菌纤溶酶基因PPFE-I,构建Pichia pastoris表达载体p PICZαA/PPFE-I,通过电击转化,p PICZαA/PPFE-I被整合到Pichia pastoris SMD1168基因组中,抗性筛选得到的阳性转化子,用终浓度为1%甲醇诱导72 h,酶活达到286 IU/ml,是野生菌的2.6倍,表达产物用SDS-PAGE进行分析,在相对分子质量63k Da处出现明显的蛋白条带,降解人血纤维蛋白试验中r PPFE-I最先降解人血纤维蛋白原的α链,其次是β链,而对γ链降解最缓慢。实现了多粘类芽孢杆菌纤溶酶基因在毕赤酵母中的表达,为植物内生菌来源溶栓药物的开发提供了新的途径。

A recombinant plasmid p ET-Dsb A / PPFE-I was constructed and used as template to amplify fibrinolytic enzyme gene PPFE-I. This was then used in Pichia pastoris expression vector p PICZαA / PPFE-I;p PICZαA / PPFE-I and integrated into the genome of Pichia pastoris SMD1168 by electroporation. Results showed that after methanol induction for 72 h,enzyme activity was 286 IU / ml. When compared to the wild type,the enzyme activity had improved 2. 6 fold. SDS-PAGE electrophoresis analysis showed that the recombinant fibrinolytic enzyme（ r PPFE-I） was expressed. In human fibrin degradation test r PPFE-I was used to firstly degrade the α chain human fibrinogen,followed by β chain,while the γ chain degradation was slowest.Endogenous Paenibacillus polymyxa fibrinolytic enzyme gene expression in Pichia pastoris was achieved,this would provide a new way to develop thrombolytic drug from endophyte sources.