杂合抗菌肽CecA-mil的改造及在毕赤酵母中的分泌表达

参照毕赤氏巴斯德酵母(Pichia pastorts)偏好密码子，改造并化学合成杂合抗菌肽CecA-mil基因，改造后的CecA-mil基因克隆到pPICZα-A载体中，构建分泌型重组酵母表达载体pPICZα-A-CM，转化Pichia pastoris受体菌X-33。在醇氧化酶(AOX)启动子调控下，分子量约1．9kD的CecA-mil杂合抗菌肽获得表达，经表达条件优化，重组酵母菌的摇瓶发酵产率可达到245μg／mL。抗菌特性研究表明，该表达产物具有广谱抗菌活性，对多数G^-菌及G^ 菌均有较好的抑菌活性，特别是对氨苄青霉素抗性菌和卡那霉素抗性菌抑杀效果更好；具有热稳定性和酸稳定性。这些特点使得重组抗菌肽CecA-mil在食品防腐、疾病防治和动物饲料添加剂等方面显露出很好的应用前景。

Modification of hybrid antimicrobial peptide CecA-mil gene and its over-secretion expression in Pichia pastoris

According to the partiality codon of Pichia pastoris, hybrid antimicrobial peptide CecA-mil gene was reconstructed, synthesized and cloned into pPICZalpha-A to construct the recombinant expression vector pPICZa-A-CM. The pPICZalpha-A-CM was transformed into yeast host strain X-33. Under the control of the promoter AOX1 (alcohol oxidase 1), a approximately 1.9 kD cecA-mil protein was expressed with the high level of 245 microg/mL after optimized the requirements for the flask-shaking culture fermentation of the Pichia pastoris rX-33/pPICZalpha-A-CM. The hybrid antibacterial peptide had a broad spectrum antibacterial activity on both gram-positive and gram-negative bacteria, especially showed potent antibacterial activity against ampicillin-resistant and kanamycin- resistant bacteria, such as Staphylococcus aureus (cowan I) and pathogenic E. coli (O1) from chicken. In addition, the hybrid antibacterial peptide showed an extreme heat-stable and acid-stable characteristic. Based on these characteristics, the recombinant antibacterial peptide CecA-mil display application foreground in the field of antisepsis of food, prevention of disease, additives of animal feedstuff and so on.