家蚕抗菌肽moricin基因的串联表达与切割产物的活性检测  

Tandem Expression of Bombyx mori Antibacterial Peptide moricin Gene and Activity Determination of Its Cleavage Product

作　　者：周雍[1] 吴程程[1] 斯洪强 李丹[1] 吕正兵[1] 盛清[1] 张耀洲[1] 聂作明[1] 

机构地区：[1]浙江理工大学生命科学学院,杭州310018

出　　处：《蚕业科学》2015年第5期864-869,共6页ACTA SERICOLOGICA SINICA

基　　金：国家级大学生创新创业训练计划项目(No.201410338015);浙江理工大学研究生创新研究项目(No.YCX12034)

摘　　要：家蚕抗菌肽是蚕体内具有免疫功能的碱性多肽,具有抑制病菌生长和杀伤癌变细胞的作用。以家蚕抗菌肽moricin为研究对象,经密码子优化,设计合成了家蚕抗菌肽moricin基因多拷贝的串联体6×moricin基因,并将其克隆到大肠杆菌原核表达载体pET-28a(+),转化E.coli BL21(DE3)感受态细胞,串联表达了融合抗菌肽6×moricin,且表达量较高。进一步通过盐酸羟胺专一性切割串联表达的融合抗菌肽6×moricin,获得相应的抗菌肽单体。体外抑菌试验验证,切割的抗菌肽moricin单体对革兰阴性菌和革兰阳性菌均有较强的抑制效果,且抑菌圈大小随抗菌肽单体浓度增加而增加。采用基因串联表达技术有望解决抗菌肽对宿主大肠杆菌的抑制作用,为利用生物反应器规模化生产家蚕抗菌肽开辟了新的技术途径。Bombyx mori antimicrobial peptides are one kind of basic polypeptide with immune function in silkworm.Bombyx mori peptides can inhibit pathogenic bacteria and kill canceration cells. In the present experiment,Bombyx mori antibacterial peptide moricin was studied. After codon optimization,the 6 multimeric moricin genes（ 6×moricin）was synthesized and then cloned into prokaryotic expression vector pET-28a（ ＋）. The recombinant plasmid was further transferred into Escherichia coli strain BL21（ DE3） competent cells to express the fusion antimicrobial peptides 6×moricin.After the highly expressed 6×moricin was cleaved specially by hydroxylamine hydrochloride,moricin monomer was obtained. Anti-bacterial test in vitro showed that the cleaved moricin monomer had strong anti-bacterial bioactivity against Gram negative bacteria and Gram positive bacteria,and the diameter of antimicrobial zones increased with moricin monomer concentration. The tandem expression method could solve the problem of inhibition of antimicrobial peptide to E. coli,and provide novel technical references to produce silkworm antimicrobial peptides in large scale using silkworm pupae as bioreactor.

关 键 词：家蚕抗菌肽 moricin 基因串联表达 抑菌活性 

分 类 号：Q78[生物学—分子生物学] S881.2[农业科学—特种经济动物饲养]