一种新型杂合抗菌肽在毕赤酵母中的表达及其抗菌活性

Expression of a Novel Hybrid Antibacterial Peptide in Pichia pastoris and Its Antibacterial Activity

目的在毕赤酵母中表达杂合抗菌肽CA(1-11)CD(12-37)ME(1-18)(ADM),并检测其抗菌活性。方法根据毕赤酵母(Pichia pastoris)偏爱密码子,合成杂合抗菌肽CA(1-11)CD(12-37)ME(1-18)基因,插入pPIC9K载体,构建重组表达质粒pPIC9K-ADM,电转化至毕赤酵母GS115,筛选阳性菌株,甲醇诱导表达,Tricine-SDS-PAGE检测ADM蛋白的表达,并对其抗菌活性进行初步检测。结果重组表达质粒pPIC9K-ADM经双酶切和测序鉴定证明构建正确;阳性酵母转化子的PCR产物可见目的基因条带;杂合抗菌肽在毕赤酵母GS115中获得分泌表达,可溶性蛋白约占菌体总蛋白的13%;且对E.coli DH5α、E.coliJM109和金黄葡萄球菌具有一定的抗菌活性。结论已在毕赤酵母GS115中表达了具有一定抗菌活性的杂合抗菌肽ADM,为相关新药的研究奠定了基础。

Objective To express hybrid antibacterial peptide CA（1-11） CD（12-37） ME（1-18）（ADM） in Pichia pastoris and determine its antibacterial activity.Methods Hybrid antibacterial peptide ADM gene was synthesized by using P.pastoris-referent codon,and inserted into vector pPIC9K.The constructed recombinant plasmid pPIC9K-ADM was transformed to P.pastoris GS115 by electron transformation,and positive recombinants were screened for expression under induction of methanol.The expressed ADM protein was identified by Tricine-SDS-PAGE and determined for antibacterial activity.Results Both restriction analysis and sequencing proved that recombinant plasmid pPIC9K-ADM was constructed correctly.Target gene fragment was amplified from the positive recombinants.Hybrid antibacterial peptide ADM was expressed in P.pastoris GS115.The expressed product in soluble form contained about 13% of total somatic protein,and showed certain antibacterial activities to E.coli DH5α,E.coli JM109 and S.aureus.Conclusion The hybrid antibacterial peptide with a certain antibacterial activity was expressed in P.pastoris GS115,which laid a foundation of development of relevant new drugs.