抗菌肽Temporin-SHf是一类具有广谱抗微生物活性的小分子多肽,该研究利用毕赤酵母表达抗菌肽Temporin-SHf。构建pPIC9K-Temporin-SHf表达载体,转化至毕赤酵母 GS115,经聚合酶链式反应(polymerase chain reaction,PCR)和反转录-聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)验证,并对获得的阳性转化子进行诱导条件优化和体外抑菌活性检测分析。结果表明,新型抗菌肽Temporin-SHf在毕赤酵母GS115中成功表达,使用甲醇终体积分数0.5%,诱导菌体起始浓度OD600为1.5,温度28 ℃,诱导72 h,分泌蛋白表达量最高,分泌表达的抗菌肽对金黄色葡萄球菌和大肠杆菌具有较强抑菌活性。该研究首次在毕赤酵母中表达新型抗菌肽Temporin-SHf,并优化了其诱导条件,为下一步工业生产提供参考。
Temporin-SHf is a kind of small molecular peptide with broad-spectrum antimicrobial activity, the expression of Temporin-SHf in Pichia pastoris could provide reference for its industrial production. The pPIC9K-Temporin-SHf expression vector was constructed and transformed into P. pastoris GS115 strain, which was then verified by PCR and RT-PCR, and the induction conditions were optimized and antibacterial activity was analyzed in vitro. The results showed that the antimicrobial peptide Temporin-SHf was expressed successfully in P. pastoris, which had antibacterial activity on Staphylococcus aureus and Escherichia coli strains. And the optimum expression conditions of Temporin-SHf were as follows: temperature 28 ℃, initial induction OD600=1.5, adding 0.5% (volume fraction) methanol, and induced for 72 h. In this study, a novel antimicrobial peptide Temporin-SHf was expressed in P. pastoris for the first time and the induction conditions were optimized, which provided theoretical base for the industrial production.
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