Molecular Cloning And Recombinant Expression Of Peroxiredoxins 6 And Thioredoxin 1 From Chinese Mitten Crab Eriocheir Sinensis

The Chinese mitten crab Eriocheir sinensis is one of the most commercially important native species for aquaculture industry in China. With the development of intensive culture and environmental deterioration, various diseases caused by bacteria, viruses and rickettsia-like organisms had frequently occurred in cultured E. sinensis populations, which resulted in enormous losses to the crab aquaculture. Like other invertebrates, E.sinensis has no memory effectors such as immunoglobulins and lymphoid cells in the immune response system, and depends on the humoral and cellular components of the innate immunity system to eliminate foreign invaders. Better understanding of the knowledge on the immune defense system of crab will be beneficial to the development of health management and diseases control in crab aquaculture.In the present study, peroxiredoxins 6 (designated EsPrx6) and thioredoxin (designated EsTrx1) were cloned from E.sinensis by using rapid amplification of cDNA ends (RACE) approaches. Quantitative real-time RT-PCR was employed to assess the mRNA expression of EsPrx6 and EsTrx1 in various tissues and their temporal expression in haemocytes of crabs challenged with Listonella anguillarum. In order to elucidate its biological functions, EsTrx1 and were recombined and expressed in E. coli BL21 (DE3).Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS). The full-length cDNA of EsPrx6 was of 1076 bp, containing a 5` untranslated region (UTR) of 69 bp, a 3`UTR of 347 bp with a poly (A) tail, and an open reading frame (ORF) of 660 bp encoding a polypeptide of 219 amino acids with the predicted molecular weight of 24 kDa. The conserved Prx domain, AhpC domain and the signature of peroxidase catalytic center identified in EsPrx6 strongly suggested that EsPrx6 belonged to the 1-Cys Prx subgroup. The mRNA transcript of EsPrx6 could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of EsPrx6 in haemocytes was down-regulated after bacterial challenge and significantly decreased compared to the control group at 12 h. As time progressed, the expression level began to increase but did not recover to the original level during the experiment. The results suggested the involvement of EsPrx6 in responses against bacterial infection and further highlighted its functional importance in the immune system of E. sinensis. The rEsPrx6 possess peroxidase activity of 23.46 U/mg protein and antioxidant activity of 14.69 U/mg. The antioxidant capacity was higher than that of GSH. All these indicated that Prx6 could function as an important antioxidant to protect crab from oxidative stress.Thioredoxin, with a redox-active disulfide/dithiol in the active site, is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. The full-length cDNA of EsTrx1 was of 641 bp, containing a 5` untranslated region (UTR) of 17 bp, a 3` UTR of 306 bp with a poly (A) tail, and an open reading frame (ORF) of 318 bp encoding a polypeptide of 105 amino acids with the predicted molecular weight of 12.2 kDa. The high similarity of EsTrx1 with Trx1s from other animals indicated that EsTrx1 should be a new member of the Trx1 sub-family. Quantitative real-time PCR analysis revealed the presence of EsTrx1 transcripts in gill, gonad, hepatopancreas, muscle, heart and haemocytes. The expression of EsTrx1 mRNA in haemocytes was up-regulated after Listonella anguillarum challenge, reached the maximum level at 6 h post stimulation, and then dropped back to the original level gradually. The rEsTrx1 was demonstrated to possess the expected redox activity in enzymatic analysis, and to be more potent than GSH in antioxidant capacity. The antioxidant capacity of rEsTrx1 was 3.06 U/mg, wich was stronger than that of GSH. The dithiol-reducing enzymatic activity (5.03) of rEsTrx1 was lower than that of Trx1 in shrimp Litopenaeus vannamei (10.44) , comparable to the specific activity of Trx1 from E. coli (4.93), calf thymus (6.50) and calf liver (5.09), and much larger than the mitochondrial Trx2 from abalone (1.83). These results together indicated that EsTrx1 could function as an important antioxidant in physiological state, and perhaps involved in the responses to bacterial challenge.