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

摘要/Abstract

摘要：

试验旨在对肉鸡Trx1和Trx2蛋白进行表达和纯化，并评价其抗氧化特性。将原核表达载体GgTrx1-pET28a（+）和GgTrx2-nsp-pET28a（+）转入大肠埃希菌中进行蛋白表达；应用镍柱亲和层析的方法对该融合蛋白进行纯化；采用胰岛素还原法对重组肉鸡Trx1和Trx2蛋白（GgTrx1和GgTrx2）进行活性鉴定；通过细胞体外试验分析比较GgTrx1和GgTrx2对大鼠肝细胞BRL-3A氧化应激保护作用的影响。结果显示，试验成功获得两种重组蛋白：GgTrx1和GgTrx2，最适诱导条件分别为37℃、0.4 mmol/L IPTG诱导4 h和37℃、1.0 mmol/L IPTG诱导4 h；纯化的重组蛋白GgTrx1和GgTrx2纯度可达到90%，浓度分别达到4.0和5.0 mg/mL。重组蛋白GgTrx1和GgTrx2均具有较高的还原胰岛素二硫键的能力，并呈现出浓度效应。体外细胞试验发现，重组蛋白GgTrx1和GgTrx2均能显著降低过氧化氢诱导的BRL-3A膜脂过氧化，保护抗氧化酶SOD和CAT活性，且重组蛋白GgTrx2效果更明显。本试验结果表明，重组蛋白GgTrx1和GgTrx2均具有生物学活性和良好的抗氧化应激特性，且线粒体型Trx2为临床治疗氧化应激性疾病提供了更多可能。

关键词: 硫氧还蛋白; 大鼠肝细胞; 过氧化氢; 原核表达; 蛋白纯化

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

中图分类号:

S858.31

余文兰, 胡莲美, 梁柏, 吴富旺, 杨帆, 郭剑英, 唐兆新. 肉鸡Trx1和Trx2蛋白的表达纯化及其抗氧化应激效果评价[J]. 《中国畜牧兽医》, 2019, 46(2): 365-372.

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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