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

摘要:

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)、Scorpion Defensin(SD)、Metalnikowin_2A和Sheep Myeloid Antibacterial Peptide(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Scorpion Defensin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。

Abstract:

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 pPICZαA-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 oxidase1)，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.