Molecular cloning and characteristic analysis of a thioredoxin from Neobenedenia melleni

Abstract

Thioredoxin (Trx) can regulate disulfide bond reduction of target proteins to maintain the reduced intracellular state in various organisms. Here, we cloned a cDNA sequence of thioredoxin from Neobenedenia melleni, which is a kind of platyhelminth parasite infecting many fishes of great economic value. The deduced N. melleni Trx (NmTrx) contained 170 amino acid residues with an active site consisting of four amino acid motif CPGC. Sequence comparison and phylogenetic tree analysis confirmed NmTrx as a distinct member of thioredoxin. Real-time quantitative polymerase chain reaction (PCR) revealed a significantly higher expression of NmTrx transcript in the adult stage compared with the egg and oncomiracidium stages. In the egg and adult stages, the NmTrx transcript level in the 32°C group was higher than those in the 18 and 25°C groups. NmTrx was expressed and purified from Escherichia coli, and antiserum against NmTrx was prepared. Western blot confirmed the higher NmTrx expression of the egg and adult stages in the 32°C group with respect to the other temperature groups. Recombinant NmTrx was able to reduce the disulfide bond in insulin, and its antioxidant capacity was determined to be 5.12 U/mg protein, similar to the classic thioredoxins. Trx activity was lower in the oncomiracidium stage and higher in the adult stage compared with the egg stage. These results indicate that NmTrx could function as an important antioxidant molecule under physiological conditions.

Keywords: Thioredoxin, Neobenedenia melleni, redox regulation, mRNA expression, prokaryotic expression