抗菌肽AD基因的改造及在毕赤酵母中的表达

Modification of Cecropin AD Gene and Its Expression in Pichia pastoris

采用PCR技术改造抗菌肽AD基因 ,将其C末端改造为Asn编码 ,改造后的抗菌肽CecropinAD基因克隆到 pPICZα_A载体上 ,构建成酵母重组分泌型表达载体 ,转化毕赤酵母 (Pichiapastoris)受体菌GS115中 .采用zerocin抗性标记筛选重组转化子 ,经摇瓶发酵 ,浓缩发酵液进行酸性聚丙烯酰胺凝胶电泳分析和抑菌活性检测 .结果表明 ,改造后的CecropinAD基因在毕赤酵母中获得了表达 ,表达产物经α_Factor信号肽引导分泌到胞外 ,具有较强的杀菌活性 .

Cecropin AD gene was amplified and modified with Asn code added to its C terminal end by PCR. The modified Cecropin AD gene was cloned into the integrative plasmid pPICZ α _A to construct a recombinant expression vector. The recombinant expression plasmid was transformed into Pichia pastoris GS115. The transformants were selected on the selection agar that contains zerocin. After fermenting and incubating, the high density fermentation product displayed antibacterial activity by testing the inhibit zone and the acidic_PAGE using E.coli K 12 D 31 as tested strain. The results showed that Cecropin AD gene was expressed in Pichia pastoris successfully. The expression product was secreted outside with the lead of α _Factor signal and had strong antibacterial activity.