Identification and expression of Fasciola gigantica thioredoxin

Abstract

In the present study, a cDNA encoding Trx from F. gigantica (FgTrx) was cloned by polymerase chain reaction (PCR). The sequence of FgTrx, analyzed by BLAST, SignalP, and ClustralW programs, showed 315 bp of an open reading frame (ORF), 12 bp 5'UTR, 78 bp 3'UTR, and the putative FgTrx peptide comprising of 104 amino acids, with a molecular weight of 11.68 kDa, with the active site containing five amino acids (tryptophan, cysteine, glycine, proline, cysteine) with a conserved dithiol motif from the two cysteines, and pI 5.86. The peptide had no signal sequence; hence, it was not a secreted protein. The recombinant FgTrx was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTrx). The FgTrx protein expression, estimated by indirect ELISA using the rabbit anti-rFgTrx as probe, showed high levels in eggs, 2- and 4-week-old juveniles, and adult parasite. In a functional test, the rFgTrx exhibited specific activity that could be suppressed by an inhibitor (PX12). When tested by immunoblotting and immunohistochemistry, rabbit anti-rFgTrx reacted with natural FgTrx at a molecular weight of 11.68 kDa from eggs, metacercariae, NEJ, 2- and 4-week-old juveniles, and adult F. gigantica. The FgTrx protein was distributed at high levels in the tegument of 2- and 4-week-old juveniles, and the tegument, parenchyma, eggs, and reproductive organs of adult parasites. FgTrx may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or drug target.

Modulation of the Functions of Goat Peripheral Blood Mononuclear Cells by Fasciola gigantica Thioredoxin Peroxidase In Vitro

“…Excretory and secretory products (ESPs), containing many active molecules, have been produced by these parasites under in vitro and in vivo conditions [ 17 ]. Previous reports showed that different molecules of ESPs from F. gigantica (FgESPs) can cause various immune responses—for example, some molecules can simulate host defense against parasites, while other have exerted the opposite effect [ 16 , 18 , 19 , 20 , 21 ]. Of particular importance to their survival is the ability of liver flukes to employ an anti–oxidant, redox-based system that protects the liver flukes against the reactive oxygen and nitrogen species produced by host immune cells and endogenous cellular metabolism [ 22 ].…”

Autonomous Non Antioxidant Roles for Fasciola hepatica Secreted Thioredoxin-1 and Peroxiredoxin-1

“…Unlike Prx1, however, novel functions of F. hepatica Trx remain to be discovered. Sequence alignments and structural models of the various F. hepatica Trxs with those from other parasites (protozoan and helminth) and mammals reveal a fully conserved structure including the protruding five amino acid motif (WCGPC) at its active site and neighbouring residues vital for its functionality ( Salazar-Calderón et al., 2001 ; Changklungmoa et al., 2014 ). This could infer that FhTrx1 exhibits some or all of the expanding assortment of endogenous and exogenous activities that are emerging for human Trx1 (hTrx).…”