CAJ | 학술논문

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pPICgk-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人酵母基因组中．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．
채용PCR방법극륭획득의혈청해항균태scygonadin기인성숙태편단，장기련접도Pichia pastoris효모표체재체pPIC9K중，구건료분비표체재체pPICgk-Scy．전격전화필적효모GS115，경표형사선화PCR감정증실료목적기인이은정정합인효모기인조중．이갑순유도표체양성극륭，상청중표체산물경과Tricine-SDS-PAGE감정여예기적목적단백대소（약10ku）상부．병이용경지공혈확산법증명료표체산물능억제혁란씨양성균화혁란씨음성균적생장．
The mature peptide sequence of antimicropeptide scygonadin in Scylla paramamosain was subcloned into Pichla pastoris expression vector pPIC9K to construct the secretory expression vector pPIC9k-Scy and subsequently transformed into the GSI15 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 I0 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.