乳铁素的融合表达及其体外抗菌活性鉴定  

Expression,Purification and in Vitro Antimicrobial Activity of Recombinant Lactoferricin

作　　者：陈紫娟[1] 金茜[1] 徐旭东[2] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009 [2]东南大学医学院,江苏南京210009

出　　处：《药物生物技术》2010年第6期478-481,共4页Pharmaceutical Biotechnology

摘　　要：为降低乳铁素Lf-cinB表达时对宿主细胞的毒害,提高乳铁素的表达量及纯化效果,合成了牛乳铁蛋白肽基因,克隆至E.coli表达载体pTYB11中,构建了与蛋白质内含子(intein)的C端融合的表达载体pTYB11-cinB。重组质粒转化至E.coliBL21(DE3)中,经IPTG诱导表达,融合蛋白intein-cinB主要以可溶形式存在于胞内。利用载体中intein中的chitin结合域,将融合蛋白通过chitin亲和层析一步纯化,经DTT诱导in-tein的自我切割活性,实现Lf-cinB在亲和柱上的切割与分离,透析冻干后得到了纯度94%的重组Lf-cinB。活性检测结果显示,重组Lf-cinB对多种检测菌均有抗菌活性。研究认为,内含肽在抗菌肽的基因工程中具有重要的应用前景。In order to reduce the harmful effect of antibacterial peptide on the host E.coli and to obtain recombinant Lf-cinB more efficiently,the bovine lactoferricin B gene,which encodes 25 amino acids was synthesized and cloned into E.coli expression vector pTYB11.The recombinant vector pTYB-cinB was transferred in E.coli BL21（DE3） cells and the Lf-cinB was expressed mainly in soluble form in fusion with an N-terminal intein tag.Then the fusion protein was purified by chitin beads column and the self-cleavage activity of the intein was induced by 1,4-dithiothreitol.After dialyzation and freeze-drying,the recombinant Lf-cinB was obtained which exhibited obvious activities against the tested bacteria.The result showed that the intein-mediated expression system will have prospective application in the production of recombinant antibacterial peptides.

关 键 词：乳铁素Lf-cinB intein融合表达 抗菌活性 

分 类 号：Q51[生物学—生物化学]