复合抗菌肽PL在毕赤酵母中的分泌表达及其活性研究

Secreted Expression of the Combinant Antimicrobial Peptide PL in Pichia pastoris and Its Antibacterial Activity in vitro

为了获得抗菌活性较强的抗菌肽,将几种抗菌肽串联起来在毕赤酵母中表达,并比较其与单独抗菌肽的抑菌活性。以GenBank中的Protegrin-1(PG-1)、ScorpionDefensin(SD)、Metalnikowin-2A和SheepMyeloidAntibacterialPeptide(SMAP-29)(序列号分别为AAB27599,AAAB27538、P80409和P49928)成熟肽段作为模板序列,根据巴斯德毕赤氏酵母(P.pastoris)偏好密码子,设计并人工合成复合抗菌肽pl基因,同时用SOE法获得ScorpionDefensin的基因,分别克隆到pPICZαA载体中,转化P.pastoris受体菌X-33,在醇氧化酶(AOX)启动子调控下,复合抗菌肽PL及SD均获得表达。体外抑菌试验检测复合抗菌肽PL与单独的蝎子防御素SD的热稳定性、酸稳定性、最低抑菌浓度等,结果显示复合抗菌肽PL及SD具有很强的热酸稳定性,而针对不同的细菌,复合抗菌肽则表现出了强于单独的SD的活性,特别是对大肠杆菌。上述结果说明了该复合抗菌肽具有很好的开发前景。

In order to obtain a high activity antibacterial peptide, An expression vector pPICZαA-pl is constructed with a tandem of four antimicrobial peptides in the same direction, which includes Protegrin-1 （PG-1）,Scorpion Defensin（ SD）, Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide （SMAP-29） （serial number in GenBank are AAB27599, AAAB27538, P80409 and P49928 respectively）. At the same time the expression vector pPICZctA-sd which express Scorpion Defensin was contructed. The expression vectors of pPICZαA-pl and pPICZαA-sd were linearized and transformed into the yeast host strain X-33 respectively. Under the control of the promoter AOX1 （alcohol oxidasel）, the peptides PL and SD were secreted expressed. Their heat-stable property,acid-stable property and MIC were detected in vitro. The results suggest the peptides PL and SD have good heat-stable and acid-stable properties, and the combinant PL peptide showes higher antibacterial activity against several Gram-positive bacteria（ G~ ） and Gram-negative bacteria（ G- ） than the peptide SD, especially against Escherichia coli. The antibacterial activity of combinant antimicrobial peptide PL shows its far exploiting perspective.