【作者】 邓文；

【导师】 肖向红；

【作者基本信息】 东北林业大学 ， 生理学， 2008， 硕士

【摘要】 抗微生物肽（Antimicrobial peptides,AMPs）作为生物体天然免疫屏障的先锋效应因子,可通过病原体诱导的免疫应答迅速合成,表现为广谱抗微生物活性,且两栖类皮肤中抗微生物肽的种类及数量最为丰富。本项目以东北地区两栖类代表物种东北林蛙（Rana dybowskii）为研究对象,构建其皮肤AMPs分泌型毕赤酵母表达系统,获得重组AMPs,进行抗菌活性鉴定,为筛选抗菌效果显著的新药候选分子以及探讨其作用机制奠定基础。方法:本实验采用PCR技术在重组pMD18-T质粒上扩增AMPs基因,在基因两端引入酶切位点,首次利用AMPs前体中天然EKR序列设计成熟肽AMPs表达程序,PCR产物经EcoRⅠ和NotⅠ双酶切,克隆入分泌型表达载体pPIC9K,PCR鉴定、DNA测序检测重组质粒,确定正确插入的重组真核表达载体。真核表达载体经LiCl转化和PEG1000转化导入毕赤酵母后涂于MD平板,挑取菌落,G418加压筛选多拷贝株,进行表型鉴定、PCR鉴定,确定获得正确插入的高拷贝重组酵母株。筛选优化重组酵母株培养条件,摇瓶培养,培养液经葡聚糖G-25层析脱盐、冻干,Bradford法检测重组AMPs含量,比浊法测定重组AMPs对标准质控G-大肠杆菌（E.coli）、G⁺金黄色葡萄球菌（Saureus）最低抑菌浓度（MIC）和协同效应。结果:（1）获得六种正确插入的重组真核分泌型表达载体pPIC9K-DB₁、pPIC9K-DB₂、pPIC9K-DB₃、pPIC9K-CS₁、pPIC9K-CS₂、pPIC9K-AM₃;并成功获得AMP-DB₂、AMP-CS₂、AMP-AM₃三种重组分泌型毕赤酵母高拷贝株,分别为5、11、1株;重组酵母摇瓶培养最优条件为0.5%甲醇,诱导36h。（2）重组AMP-DB₂、AMP-CS₂、AMP-AM₃对G⁺S.aureus的MIC分别为7.44、14.88、14.88（mg/ml）,而对Ecoli的MIC则分别为59.52、29.76、59.52（mg/ml）;AMP-DB₂与AMP-CS₂的组合对G^-E.coli的MIC比单独作用时分别降低至75%和50%,但重组AMPs对G⁺S.aureus无明显协同作用。结论:本项目首次利用AMPs前体中天然EKR序列设计成熟肽AMPs表达载体,建立了东北林蛙皮肤成熟AMPs的分泌型毕赤酵母表达系统。并通过重组酵母诱导成功生成对G⁺和G^-菌有抑杀作用的重组AMPs,对G^-E.coli活性的抑制有明显协同作用。更多还原

【Abstract】 Antimicrobial peptides（AMPs）which are pioneer effectors in natural immunity defense line,synthesize rapidly through the immune response inducted by pathogen.The AMPs has broad-spectrum antimicrobial activity.The type and quantity of AMPs from the amphibian skin and the secretion are richest in all Organisms.We use the Pichia pastoris expression system to express several AMPs from the skin of Rana dybowskii which is one of main amphibian species in northeast of china,they could become the experimental referece for the production of a new speciall antimicrobial drug,and be used to research the mechanism of antimicrobial effect and synergetic effect.Methods:Using the six recombinant pMD18-T as the template,six AMPs genes were amplified with PCR and double digested with EcoRⅠand NotⅠ.These genes were ligated into vector pPIC9K digested with EcoRⅠand NotⅠ.Then they were transformed into host strain GS115,several His+cell line was screened and multicopy transformants were screened by various G418 concentrations.The transformants were cultivated in flasks,and firstly used the nature EKR sequence in AMPs Precursors to get mature peptides in culture medium.The supernatant were desalted by Sephadex G-25,and then lyophilized.We tested minimal inhibitory concentrations（MICs）of AMPs on G⁺（Escherichia coli）、G^-（Staphyloccocus aureus） in vitro,and they are combinated with Each other for the test of synergetic effect.The results were as follows:（1）we got six recombinant expression plasmids pPIC9K-DB₁、pPIC9K-DB₂、pPIC9K-DB₃、pPIC9K-CS₁、pPIC9K-CS₂、pPIC9K-AM₃,and got three recombined P.pastoris cell line which can successfully express AMP-DB₂、AMP-CS₂、AMP-AM3,respectively are five、eleven one transformants.The optimum condition is inducted by 0.5%methanol for 36 hour.（2）The antimicrobial effect on G⁺S.aureus of all AMPs is better than G^-E.coli,and the average MIC of G⁺S.aureus is 25%of G’E.coli.The mixture of AMP-DB₂ and AMP-CS₂can suppress G⁺E.coli better,the MIC is reduced by 75% and 50%respectively.The synergetic effect of AMPs on S.aureus is inapparent.Conclutions:This study has constructed P.pastoris expression system of AMPs from the skin of Rana dybowskii.The recombined AMPs have antimicrobial activity,and some of them displayed synergetic effect on G^-E.coli.更多还原