布氏锥虫Trx-putative和Trx-like的抗氧化功能研究

Analysis of Antioxidant Function of Trypanosoma brucei Trx-putative and Trx-like

硫氧还蛋白(Trx)是一种氧化还原调节蛋白,参与锥虫抗宿主氧化,生物信息学分析发现Trx-putative和Trx-like含有与Trx一样的“Tioredoxin-like super family”结构域,为了阐明其抗氧化功能,通过原核表达方法获得其重组蛋白,重组蛋白免疫SD大鼠制备抗性血清,间接免疫荧光试验观察其在虫体内的表达情况,DTT/胰岛素还原法测定其二硫键异构酶活性,金属催化氧化法测定其体外保护DNA超螺旋活性,利用PCR点突变的方法分析其结构与功能的关系。结果显示,获得了纯度较高的重组蛋白,Trx-putative和Trx-like广泛分布于虫体胞质内;Trx-putative可以催化胰岛素二硫键的打开,而Trx-like不具备此功能;Trx-putative和Trx-like均可对氧化损伤的pUC19质粒产生保护,突变蛋白的二硫键异构酶活性显著降低,抗氧化作用减弱。这些结果提示布氏锥虫Trx-putative和Trx-like具有抗氧化功能。

Thioredoxin(Trx)is a redox regulatory protein that is involved in anti-host oxidation of trypanosomes.It is found that Trx-putative and Trx-like contain the same"tioredoxin-like super family"domain as Trx.In order to clarify its antioxidant function,the recombinant proteins were obtained by prokaryotic expression method.Resistant serum was prepared by immunizing SD rats with recombinant protein.IFA was used to observe its expression in Trypanosoma brucei,and DTT/insulin reduction method was used to determine its disulfide isomerase activity,in vitro protective DNA superhelix activity was determined by metal catalytic oxidation method,and the relationship between structure and function was analyzed by PCR point mutation method.The results showed that the recombinant protein with high purity was obtained.Trx-putative and Trx-like are widely distributed in the cytoplasm of Trypanosoma brucei.Trx-putative can catalyze the opening of insulin disulfide bond,while Trx-like does not have this function.Both Trx-putative and Trx-like can protect PUC19 plasmid from oxidative damage.The disulfide isomerase activity of the mutant protein was significantly reduced,and the antioxidant activity was weakened.The results suggest Trx-putative and Trx-like had antioxidant function.