作　　者： ; ; ; ; ; ;

机构地区： 华南农业大学兽医学院

出　　处： 《中国兽医科学》 2015年第4期429-434,共6页

摘　　要： 为了研究肉鸡线粒体型硫氧还蛋白(GgTrx2)的功能,提取肉鸡肝总RNA,经RT-PCR扩增去除信号肽序列的GgTrx2基因,构建原核表达载体pET-28a(+)-GgTrx2,转化入大肠杆菌BL21(DE3),IPTG诱导表达;以Ni-NTA纯化重组蛋白并评价其活性;分析GgTrx2对脂多糖(LPS)诱导的鸡肝细胞氧化应激的影响。结果显示,构建的原核表达载体pET-28a(+)-GgTrx2在37℃经1.0mmol/L IPTG诱导4h蛋白表达效果最好,经Ni-NTA柱纯化后纯度达90%以上,质量浓度为5.0mg/mL。该重组蛋白具有较高的还原胰岛素二硫键的能力,并呈现浓度效应。抗氧化功能分析表明,GgTrx2预处理能显著降低LPS诱导的鸡肝细胞膜脂过氧化和保护抗氧化酶SOD和CAT的活性。结果表明,本研究原核表达的GgTrx2蛋白具有良好的生物学活性和抗氧化应激特性,为进一步阐明其生物学功能和用于治疗氧化应激性疾病奠定了基础。 The objective of this study was to clarify the biological function of broiler chicken(Gallus gallus)mitochondrial thioredoxin-2(GgTrx2).The GgTrx2 cDNA without signal peptide was amplified by RT-PCR with the total RNA of specific pathogen free Yellow-Feathered broiler chicken as template.The recombinant plasmid pET-28a(+)-GgTrx2 was constructed,then transformed to Escherichia coli BL21(DE3),and induced to express GgTrx2 with IPTG.After purification of GgTrx2 by Ni-NAT chromatography,the activity was determined by insulin assay.The protective effect of GgTrx2 on oxidative stress induced by lipopolysaccharide(LPS)in chicken hepatocyte cell line was also evaluated.The results showed that GgTrx2 was induced optimally to express with 1.0mmol/L IPTG for 4hat 37℃.The recombinant GgTrx2 protein was purified with a purity of more than 90% and a concentration of 5.0mg/mL,which possessed good disulfide reductase activity in a concentration-dependent manner.Furthermore,GgTrx2 pretreatment significantly alleviated oxidative stress induced by LPS in chicken hepatocytes,with attenuated lipid peroxidation and increased activities of superoxide dismutase and catalase.Overall,the results suggest that the prokaryotically expressed GgTrx2 exhibited good biological activity and protective effect on LPSinduced oxidative stress,which laid the foundation for the comprehensive studies of its biological function and the therapy of oxidative stress-related diseases.

关 键 词： 肉鸡 线粒体型硫氧还蛋白 原核表达 蛋白纯化

领　　域： [农业科学] [农业科学]