天蚕抗菌肽B在毕赤酵母中的表达

Expression of cecropin B in Pichia pastoris

Cecropins B是一种昆虫抗菌肽,具有分子量小,热稳定性、水溶性好、抗菌谱广等优点,更为重要的是抗菌肽对真核细胞几乎没有作用,仅仅作用于原核细胞和发生病变的真核细胞,是目前已知天然抗菌肽中活力最强的一种。根据毕赤氏巴斯德酵母(Pichia pastoris)偏好密码子,改造并化学合成天蚕素基因B,克隆到pPIC9K载体中,构建分泌型重组酵母表达载体CB-pPIC9K,转化Pichia pastoris受体菌KM71,在醇氧化酶(AOX)启动子调控下,Cecropin B获得表达,分子量约3.8KD,摇瓶发酵产率可达到200μg/mL,并对其抗菌活性进行了初步测定。结果表明我们表达的重组Cecropins B对G-菌有较好的抑菌活性,且具有较好的热稳定性。这些特点使得重组抗菌肽Cecropins B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。

Cecropins B is a kind of insect antibacterial peptide, which have the lower molecular weight , thermostability, water-solubility good and have bactericidal activity against a broad range of bacteria. Especially, nearly no have the function to the eukaryotic cell . At present, cecropins B is the activities strongest one kind in which is known nature＇s antibacterial peptide. According to the partiality codon of Pichia pastoris, antibacterial peptides cecropin B gene was reconstructed, synthesized and cloned into pPIC9K to construct the recombinant expression vector CB-pPIC9K. The CB- pPIC9K was transformed into P. pastor/s KM71. Under the control of the promoter AOX, cecropinB protein was expressed with the high level of 200μg/mL in the flask-shaking culture fermentation. In addition, the cecropinB protein with antibacterial activity was analyzed. The results indicated which the cecropins B has the good antibactrial activities to grem-negative bacterial and the thermostability. These characteristics of cecropins B display very good application prospect in the field of antisepsis of food anticorrosion, disease preventing and additives of animal feedstuff and so on.