拟穴青蟹抗菌肽scygonadin在毕赤酵母中的表达及其抗菌活性

Abstract

采用PCr方法克隆获得拟穴青蟹抗菌肽SCygOnAdIn基因成熟肽片段,将其连接到PICHIA PASTOrIS酵母表达载体PPIC9k中,构建了分泌表达载体PPIC9k-SCy.电击转化毕赤酵母gS115,经表型筛选和PCr鉴定证实了目的基因已稳定整合入酵母基因组中.以甲醇诱导表达阳性克隆,上清中表达产物经过TrICInE-SdS-PAgE鉴定与预期的目的蛋白大小(约10ku)相符.并利用琼脂孔穴扩散法证明了表达产物能抑制革兰氏阳性菌和革兰氏阴性菌的生长.

The mature peptide sequence of antimicropeptide scygonadin in Scylla paramamosain was subcloned into Pichia pastoris expression vector pPIC9K to construct the secretory expression vector pPIC9k-Scy and subsequently transformed into the GS115 yeast strain using the electroporation.The positive clones,of which the chromosomes were integrated with scygonadin gene,were identified using the phenotype screning and PCR.Then they were induced by methanol and the expression product in the supernatant of about 10 ku,as preriously predicted was examined using Tricine-SDS-PAGE.Results of agarose diffusion assay indicate that the expressed scygonadin exhibits the antibacterial activity against both Gram negative bacteria and Gram positive bacteria.