人源抗菌肽AP-57的原核表达与纯化  

Expression and Purification of Antimicrobial Peptides AP-57

作　　者：马先君 杨梅佳 仝爱平[1] 

机构地区：[1]四川大学生物治疗国家重点实验室,成都610000

出　　处：《科学技术与工程》2016年第23期248-252,共5页Science Technology and Engineering

基　　金：国家自然科学基金项目(31471286)资助

摘　　要：构建AP-57/C10orf99重组表达载体,并在BL21中进行重组蛋白的表达。经纯化获得高纯度的AP-57,从而为其功能性研究提供基础。通过合成AP-57基因序列,连接入p ET32质粒中与Trx标签和His标签融合,转化进DH5α,经筛选、鉴定为阳性克隆菌落中提取的质粒转入基因工程化BL21中进行表达。通过优化表达条件如温度、诱导时间和诱导剂（IPTG）浓度来提高重组蛋白表达量。最后,通过亲和层析浓缩获得融合蛋白,去标签和阳离子交换层析、脱盐步骤获得纯的AP-57蛋白。结果是成功构建了AP-57的诱导表达和纯化体系：当菌液OD为0.6~0.8时,用0.5 mmol/LIPTG,25℃,诱导培养6 h;通过纯化后获得的样品经质谱和SDS-PAGE鉴定为AP-57且含一个链内二硫键。实验建立了一条完整的AP-57制备工艺,为后续其功能性研究奠定了基础。Construction of AP-57 /C10orf99 expression vector and expression of recombinant proteins in the BL21,and then through purification obtain the samples of high-purity AP-57,so as a basis for functional study was provided. Synthesis of AP-57 by gene sequences,and connections into the p ET32 plasmid integration with the Trx and His tags,transformed into DH5α,after screening and identification for colonies of clones to extract grain BL21 expressed. Optimizing expression conditions such as temperature,time and inducers( IPTG) concentration to improve the expression of recombinant protein; and,finally,get fusion proteins by affinity chromatography enrichment,remove label,the cation exchange chromatography,and desalination steps to obtain the pure AP-57. The result has been to build an AP-57 induced expression and purification system: when the bacteria liquid OD is 0. 6 ~0. 8,with 0. 5 mmol / LIPTG,25 ℃,induction culture 6 h; Through the purified samples via mass spectrometry and SDS-PAGE electrophoresis appraisal for AP-57,and AP-57 containing a disulfide bond in the chain. This study established a complete AP-57 preparation technology,laid the foundation for subsequent its functional study.

关 键 词：AP-57/C10orf99 原核表达 蛋白纯化 

分 类 号：TQ936.16[化学工程] Q815[生物学—生物工程]