杂合抗菌肽Mel-MytB在毕赤酵母中的表达及其抗菌活性测定

为了获得抗菌活性较强的抗菌肽，将蜂毒肽（Melittin）与贻贝素B （Mytilin-B）的核心功能序列杂合，以Melittin （3-14）和Mytilin-B （13-27）的成熟肽段作为模板序列，根据巴斯德毕赤酵母（Pichia pastoris）偏好密码子，采用SOE方法合成杂合抗菌肽Mel-MytB（MEM）基因。改造后的基因克隆到pPICZα-A质粒，构建分泌型重组酵母表达载体pPICZα-A-MEM，而后经Sac Ⅰ酶切线性化后电转入毕赤酵母受体菌X-33。结果表明，在醇氧化酶（AOX）启动子调控下，分子质量约3.0 ku的Mel-MytB杂合抗菌肽获得表达，具有热稳定性和酸稳定性，煮沸40 min、pH 2.0~10.0范围内抑菌活性基本不变。抗菌特性研究结果表明，该表达产物具有广谱抗菌活性，其对大肠杆菌ATCC25922、金黄色葡萄球菌ATCC25923/ATCC6538、肠道沙门氏菌ATCC13076、鼠伤寒沙门氏菌ATCC14028、副溶血弧菌ATCC17802、创伤弧菌ATCC27562、枯草芽孢杆菌ATCC6633的MIC值分别为5.1、2.2、2.0、4.6、7.6、9.4、13.2和59.4 μg/mL。因此，重组抗菌肽Mel-MytB在疾病防治和动物饲料添加剂等方面具备较好的应用前景。

Expression of Hybrid Antimicrobial Peptide Mel-MytB in Pichia pastoris and Detection of Its Antibacterial Activity

In order to abtain a high activity antimicrobial peptide,an expression vector was constructed with a tandem of Melittin and Mytilin-B.According to the partiality codon of Pichia pastoris,the Melittin(3-14)-Mytilin-B(13-27) hybrid antimicrobial peptide,Mel-MytB (MEM) gene was synthesized by SOE method,and cloned into pPICZa-A to construct the recombinant expression vector pPICZa-A-MEM.pPICZα-A-MEM was linearized by Sac Ⅰ and electroporated into Pichia pastoris X-33.The results showed that under the control of the promoter alcohol oxidase (AOX),an approximately 3.0 ku Mel-MytB protein was expressed,which had thermal stability and acid stability.The antibacterial activity was basically unchanged after boiling for 40 min and pH 2.0-10.0.Antibacterial assays demonstrated that Mel-MytB had broad-spectrum antibacterial activity.The MIC to Escherichia coli ATCC25922,Staphylococcus aureus ATCC25923,Staphylococcus aureus ATCC6538,Salmonella enterica ATCC13076,Salmonella Typhimurium ATCC14028,Vibrio parahaemolyticus ATCC17802,Vibrio vulnificus ATCC27562 and Bacillus subtilis ATCC6633 were 5.1,2.2,2.0,4.6,7.6,9.4,13.2 and 59.4 μg/mL,respectively.Therefore,the recombinant antimicrobial peptide Mel-MytB had a good application prospect in disease prevention and animal feed additives.