Cheng Shixiong, Li Chenghua, Wang Yi, Yang Limeng, Chang Yaqing
Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, PR China.
Key Laboratory of Mariculture & Stock Enhancement in North China's Sea, Ministry of Agriculture, Dalian Ocean University, Dalian 116023, PR China; School of Marine Sciences, Ningbo University, Ningbo, Zhejiang Province 315211, PR China.
Fish Shellfish Immunol. 2016 Nov;58:165-173. doi: 10.1016/j.fsi.2016.08.061. Epub 2016 Sep 14.
As the most important disulfide bond reducates of intracellular oxidordeuctase, thioredoxin (TRX) plays a crucial role in maintaining reducing state of intracellular proteins to normally perform their function. In this study, a cDNA of TRX-like protein gene from Apostichopus japonicus (denoted as AjTRX) was cloned and characterized. The full-length cDNA of AjTRXwas of 1870 bp, consisting of a 5'-UTR of 101 bp, a long 3'-UTR of 887 bp and a 882 bp open reading frame (ORF) encoding a 293 amino acids. The predicted molecular mass and the theoretical PI of the deduced amino acids of AjTRX were 32.3 kDa and 5.52, respectively. Phylogenetic trees showed that AjTRX had a closer evolution relationship with TRX from Strongylocentrotus purpuratus. AjTRX was found to be ubiquitously expressed in all examined tissues including longitudinal muscle, coelomocytes, tube feet, intestine, respiratory tree and body wall indicating a general role in physiological processes. Temporal expression pattern of AjTRX in coelomocytes showed that AjTRX reached two peak expression levels at 8 h and 48 h after Vibrio splendidus challenge with a 8.6 and 9.3-fold increase compared to their control groups, respectively. The recombinant AjTRX protein (rAjTRX) displayed obvious antioxidant activity in a dose-dependent manner, and the higher reducing activity was detected in 20 μM experimental group. All these results strongly suggested that AjTRX could play an important role as an antioxidant in a physiological context, and might be involved in the process of bacterial challenge.
作为细胞内氧化还原酶最重要的二硫键还原剂，硫氧还蛋白（TRX）在维持细胞内蛋白质的还原状态以使其正常发挥功能方面起着至关重要的作用。在本研究中，克隆并鉴定了仿刺参硫氧还蛋白样蛋白基因（命名为AjTRX）的cDNA。AjTRX的全长cDNA为1870 bp，由101 bp的5'-UTR、887 bp的长3'-UTR和一个882 bp的开放阅读框（ORF）组成，该开放阅读框编码293个氨基酸。预测的AjTRX推导氨基酸的分子量和理论pI分别为32.3 kDa和5.52。系统发育树显示，AjTRX与紫海胆的TRX具有更密切的进化关系。发现AjTRX在所有检测的组织中普遍表达，包括纵肌、体腔细胞、管足、肠、呼吸树和体壁，表明其在生理过程中具有普遍作用。AjTRX在体腔细胞中的时间表达模式表明，在灿烂弧菌攻击后8小时和48小时，AjTRX达到两个峰值表达水平，与对照组相比分别增加了8.6倍和9.3倍。重组AjTRX蛋白（rAjTRX）以剂量依赖的方式表现出明显的抗氧化活性，在20 μM实验组中检测到较高的还原活性。所有这些结果强烈表明，AjTRX在生理环境中可能作为一种抗氧化剂发挥重要作用，并且可能参与细菌攻击过程。
