海蚕新型抗菌肽Perinerin在毕赤酵母中的表达及活性测定

摘要：利用巴斯德毕赤酵母表达系统，对海蚕抗菌肽 Perinerin 进行分泌性表达，并进行活性检测。首先通过 SOE 法（Gene splicing by over lap extension）设计三对互补引物来合成完整的Perinerin 的基因,将该基因片断插入到含有AOX1启动子和α分泌信号肽序列载体pPICZαA中,构建了重组表达质粒 pPICZαA-PEN ,转化Pichia pastoris GS115 宿主菌,利用甲醇来诱导外源基因在阳性菌株中的表达,表达产物经 Tricine-SDS-PAGE 电泳验证。生物学活性检测证实重组蛋白对部分革兰阳性及革兰阴性细菌有明显抑制作用,尤其对绿脓杆菌有强烈的抑制作用。

Expression and Bioactivity Assay of a Novel Clamworm Antimicrobial Peptide Perinerin in Pichia pastoris

Abstract: To obtain large amounts of active perinerin and characterize its main physiochemical features, we expressed perinerin in Pichia pastoris. Intact Perinerin gene amplified by the gene SOEing method（Gene splicing by overlap extension）was cloned into expression vector pPICZαA and obtained recombinant vector pPICZαA-PEN, then pPICZαA-PEN was expressed in the Pichia pastoris GS115,The expressed sample was analyzed by Tricine-SDS-PAGE, Our results showed that Pichia pastoris was a suitable system producing the secreted form of Perinerin. Bioactivity assay showed that the recombinant Perinerin had marked antimicrobial effects.