无标签重组人硫氧还蛋白的大规模表达、纯化及活性检测

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.

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

Objective:To construct prokaryotic expression system for mass production of recombinant human thioredoxin and establish the purification process of thioredoxin.Methods: Total RNA was extracted from HEK293(human embryonic kidney cells).The thioredoxin coding sequence 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/5L 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.