肉鸡Trx1和Trx2蛋白的表达纯化及其抗氧化应激效果评价

doi:10.16431/j.cnki.1671-7236.2019.02.005

Abstract

Abstract:

The aim of this experiment was to express and purify Trx1 and Trx2 proteins and evaluate their antioxidant properties.The constructed recombinant plasmid GgTrx1-pET28a(+) and GgTrx2-nsp-pET28a(+) were transformed into E.coli for expression under induction of IPTG and purified by Ni-NAT chromatography.The recombinant proteins GgTrx1 and GgTrx2 activity were determined by insulin assay.The protective effect of GgTrx1 and GgTrx2 on oxidative stress induced by hydrogen peroxide (H₂O₂) in BRL-3A cell line was also evaluated.The results showed that the two recombinant proteins GgTrx1 and GgTrx2 were successfully obtained,and the optimal induction conditions were 37℃,0.4 mmol/L IPTG induction for 4 h and 37℃,1.0 mmol/L IPTG for 4 h,respectively.The recombinant proteins GgTrx1 and GgTrx2 were purified with a purity of more than 90% with concentration of 4.0 and 5.0 mg/mL, respectively.The purified recombinant proteins GgTrx1 and GgTrx2 both had higher ability to reduce insulin disulfide bonds in a concentration-dependent manner.In vitro cell assays showed that both GgTrx1 and GgTrx2 significantly reduced hydrogen peroxide-induced BRL-3A membrane lipid peroxidation and protected antioxidant enzymes SOD and CAT activities,and GgTrx2 was more effective.The results of this experiment indicated that both purified GgTrx1 and GgTrx2 had biological activity and good anti-oxidative stress characteristics,and mitochondrial Trx2 provided more possibilities for clinical treatment of oxidative stress diseases.

Key words: thioredoxin; rat liver cell; hydrogen peroxide; prokaryotic expression; protein purification

CLC Number:

S858.31

YU Wenlan, HU Lianmei, LIANG Bai, WU Fuwang, YANG Fan, GUO Jianying, TANG Zhaoxin. Expression and Purification of Trx1 and Trx2 Proteins and Evaluation of Their Antioxidative Stress[J]. , 2019, 46(2): 365-372.

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Expression and Purification of Trx1 and Trx2 Proteins and Evaluation of Their Antioxidative Stress

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