杂合抗菌肽MgJ基因在毕赤酵母中表达条件的优化

Optimization of Expression Conditions for Hybrid Antimicrobial Peptide Mg J in Pichia pastoris

为确定杂合抗菌肽MgJ基因的最佳诱导表达条件,将构建好的重组表达载体p PICZαA-MgJ经SacⅠ线性化处理,电转入巴斯德毕赤酵母GS115,利用抗生素Zeocin筛选阳性克隆,聚合酶链式反应(polymerase chain reaction,PCR)扩增验证,甲醇诱导表达。优化杂合抗菌肽MgJ诱导剂浓度、诱导时间、诱导温度、初始菌体浓度表达条件。结果表明杂合抗菌肽MgJ的最佳表达条件为:28℃、250 r/min诱导培养,每24 h添加体积分数0.5%的甲醇,诱导时间120 h,MgJ表达量可达11.9 mg/L。纯化后获得的表达产物MgJ对S.aureus ATCC 29213有较好的抑菌活性,最小抑菌浓度(minimal inhibitory concentration,MIC)为10μg/m L。

The objective of this study was to determine the optimal induction conditions for the expression of hybrid antimicrobial peptide MgJ in Pichia pastoris.The Sac I-linearized recombinant expression vector p PICZαA-MgJ was transformed into Pichia pastoris GS115 by electroporation.The positive clones were screened with Zeocin and identified by PCR.The protein expression was induced by methanol and the expression conditions of hybrid antimicrobial peptide MgJ were optimized.The results showed that the optimum expression conditions of MgJ were as follows:28 ℃,250 r/min,adding 0.5%(V/V) methanol to the culture every 24 h,and induced expression time 120 h.The amount of expressed MgJ could reach up to 11.9 mg/L.After purification,the expressed product MgJ had better antimicrobial activity on S.aureus ATCC 29213 with a minimal inhibitory concentration(MIC) of 10 μg/m L.