人源抗菌肽LL-37及其改良体串联体的原核表达及纯化  

Prokaryotic expression and purification of gene multimers from human antimicrobial peptide LL-37 and its variant based on sequence reformation

作　　者：杨浩[1] 姚海燕[1] 陈圆圆 阎波[1] 郭鹏[1] 马驰[1] 韩跃武[1] 

机构地区：[1]兰州大学基础医学院生物化学与分子生物学研究所,兰州730000 [2]甘肃省平凉市中医院,甘肃平凉744000

出　　处：《中国生物制品学杂志》2012年第5期570-573,共4页Chinese Journal of Biologicals

摘　　要：目的克隆人源抗菌肽(Antimicrobial peptide,AMP)LL-37及其改良体串联体基因,原核表达并纯化重组蛋白。方法利用生物信息学手段,对人源抗菌肽LL-37基因序列进行改良设计,合成人源抗菌肽LL-37及其改良体基因片段,经重叠延伸PCR获得人源抗菌肽LL-37及其改良体串联体基因,克隆至原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-dLL-37,转化大肠杆菌BL21(DE3)pLysS,IPTG诱导表达,Tricine SDS-PAGE分析重组蛋白的表达形式,镍金属螯合亲和层析纯化重组蛋白。结果改良后的人源抗菌肽LL-37的等电点、稳定性及其在大肠杆菌中的半衰期均显著提高;重组表达质粒pET-28a-dLL-37经测序鉴定构建正确;表达的重组蛋白相对分子质量约为11 000,主要以包涵体形式存在;纯化的重组蛋白纯度可达95%,浓度为0.473 mg/ml。结论已成功在大肠杆菌中表达并纯化了人源抗菌肽LL-37及其改良体串联体,为其后续生物学活性的研究奠定了基础。Objective To clone the gene multimers from human antimicrobial peptide（AMP） LL-37 and its variant,express in prokaryotic cells and purify the expressed product.Methods The variant of human AMP LL-37 was designed by bioinformatic tool,based on which AMP LL-37 and its variant were synthesized.The gene multimers were amplified by overlap PCR and cloned into prokaryotic expression vector pET-28a（＋）,and the constructed recombinant plasmid pET-28a-dLL-37 was transformed to E.coli BL21（DE3） pLysS and induced with IPTG.The expressed recombinant protein was analyzed by Tricine SDS-PAGE and purified by nickel ion affinity chromatography.Results As compared with those of AMP LL-37,the isoelectric point,stability and half-life in E.coli of the gene multimers increased significantly.Sequencing result proved that recombinant plasmid pET-28a-dLL-37 was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 11 000,mainly existed in a form of inclusion body,and reached a purity of 95% and a concentration of 0.473 mg / ml after purification.Conclusion The gene multimers from human AMP LL-37 and its variant were successfully expressed in E.coli and purified,which laid a foundation of further study on its biological activity.

关 键 词：抗菌肽 串联体 重叠延伸PCR 原核细胞 基因表达 纯化 

分 类 号：Q786[生物学—分子生物学]