无标签重组人硫氧还蛋白的大规模表达、纯化及活性检测  

Expression,Purification and Characterization of Non-taged Recombinant Human Thioredoxin

作　　者：郭云萍[1] 孙璐[1] 张立剑[1] 王增禄[2] 高超[1] 杨强[1] 刘毅[1] 张英起[2] 屈延[1] 陶凌[1] 

机构地区：[1]第四军医大学西京医院,西安710032 [2]第四军医大学药学系生物技术中心,西安710032

出　　处：《中国生物工程杂志》2012年第8期62-67,共6页China Biotechnology

基　　金：国家科技重大专项(2009ZXJ09004-088);国家自然科学基金(81070676);国家重大新药创制计划(2012ZX09J12108-06B)资助项目

摘　　要：目的:利用基因工程的方法原核表达无标签的重组人硫氧还蛋白(rhTrx)并对其进行大规模表达、纯化和鉴定。方法:从人胚胎肾HEK293细胞中提取总RNA,反转录合成cDNA,经PCR扩增、酶切后连入pET-22b(+)载体构建重组质粒,重组质粒转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达,经两步离子交换层析纯化重组蛋白,采用SDS-PAGE、Western blotting、HPLC、MALDI-TOF-MS及经典的胰岛素二硫键还原法对重组蛋白进行鉴定。结果:构建成功了rhTrx基因表达载体;实现了rhTrx在原核细胞中的可溶性表达;纯化出的蛋白经SDS-PAGE和Western blotting分析证实为rhTrx;HPLC和MALDI-TOF-MS分析表明,纯化出的目的蛋白纯度大于95%;胰岛素二硫键还原法证实纯化出的rhTrx具有生物学活性。结论:成功构建了rhTrx的原核表达体系,建立了rhTrx的纯化和鉴定方法,为其进一步的理论研究和生产开发提供了有效基础数据。Objective ：To construct prokaryotic exp thioredoxin and establish the purification process of ression system for mass thioredoxin. Methods ： HEK293 （human embryonic kidney cells）. The thioredoxin coding sequence production of recombinant human Total RNA was extracted from was subcloned into the pET-22b （ ＋ ） vector after amplified by PCR. The recombinant plasmids were transformed into E. coli BL21 （ DE3 ） , and the thioredoxin was expressed with IPTG induction. The expressed thioredoxin was purified by two-step ion exchange chromatography and tested by SDS-PAGE, Western blotting, MALDI-TOF-M, HPLC, and insulin disulfide reduction assay for identification, purity assay and activity determination, respectively. Results： Gene sequencing demonstrated that thioredoxin coding sequence was cloned into pET-22b （ ＋ ） vector successfully. The prokaryotic expression system achieved high yield of thioredoxin （180 mg/SL of fermentation broth）, which was identified by Western blotting and MALDI-TOF-MS, with an estimated the molecular weight of 12 000. The purity of thioredoxin is more than 95 %. The activity of purified thioredoxin had the same activity as the standard control. Conclusion： The prokaryotic expression system could achieve mass production of recombinant human thioredoxin, which can be highly purified by two-steps ion exchange chromatography. This preliminary study provides the foundation for the large-scale industrial production of thioredoxin.

关 键 词：重组人硫氧还蛋白(rhTrx) 表达纯化 生物活性 

分 类 号：Q786[生物学—分子生物学]