抗菌肽magainin Ⅱ的密码子优化及在大肠杆菌和毕赤酵母中的表达  

Optimization of coding sequences and expression of antimicrobial peptide magainin Ⅱ in Escherichia coli and Pichia pastoris

作　　者：陈玉海[1] 陈庆煌[2] 陈科[1] 章亭洲[3] 陈吉龙[1,2] 

机构地区：[1]中国科学院微生物研究所中国科学院病原微生物与免疫学重点实验室,北京100101 [2]福建农林大学动物科学学院动物医学系,福建福州350002 [3]浙江工商大学环境科学与工程学院,浙江杭州310035

出　　处：《生物工程学报》2014年第4期615-624,共10页Chinese Journal of Biotechnology

基　　金：国家科技重大专项"传染病重大专项"(No.2013ZX10004-611)资助~~

摘　　要：Magainin Ⅱ是非洲爪蟾皮肤分泌的一种抗菌肽,在低浓度下即能够对细菌、真菌、原虫、肿瘤等都具有杀伤作用,并且与同是来源于非洲爪蟾皮肤的另一种抗菌肽PGLa在杀菌、抗肿瘤等方面具有很好的协同效应。文中分别按照大肠杆菌和毕赤酵母的密码子利用频率,对抗菌肽magainin Ⅱ以及杂合抗菌肽magainin Ⅱ-PGLa,进行了密码子优化。构建了高效表达抗菌肽magainin Ⅱ的原核表达载体,经过蛋白酶切割获得纯化的抗菌肽magainin Ⅱ。同时进一步构建了杂合抗菌肽magainin Ⅱ-PGLa的分泌型毕赤酵母表达载体,实现了杂合抗菌肽在毕赤酵母中的分泌表达。本研究为magainin Ⅱ和magainin Ⅱ-PGLa的生产与应用奠定了基础。The antimicrobial peptide magainin Ⅱ is expressed in the skin of the African clawed frog, Xenopus laevis, and exhibits a broad spectrum of antimicrobial activity as well as tumoricidal properties at low concentrations. In addition, magaininⅡ plays a synergistic role during antimicrobial and tumoricidal processes with another antimicrobial peptide PGLa that is also expressed inXenopus laevis. The optimized cDNA sequence of magaininⅡ and magaininⅡ -PGLa hybrid peptide according to E. coli or Pichia pastoris codon usage frequency were synthesized and sub-cloned into prokaryotic expression vector pGEX and Pichia pastoris secreted expression vector pPIC9k. The resulting recombinant plasmids were named as pGEX-magainin Ⅱand pPIC9k-magainin Ⅱ -PGLa. The GST-magainin Ⅱfusion protein was highly expressed in E. coli. Furthermore, magainin Ⅱ was successfully purified by digestion with PreScission Protease to cleave the GST tag. Additionally, our data obtained from the ELISA revealed that magainin Ⅱ-PGLa hybrid peptide was successfully expressed in Pichia pastoris. These experiments establish a useful system for further studies of these antimicrobial peptides.

关 键 词：抗菌肽 magaininⅡ 大肠杆菌 毕赤酵母 密码子利用频率 

分 类 号：Q78[生物学—分子生物学]