title: Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase.
abstract: The Fasciola gigantica thioredoxin-glutathione reductase (FgTGR) gene is a fusion between thioredoxin reductase (TR) and a glutaredoxin (Grx) gene. FgTGR was cloned by polymerase chain reaction (PCR) from adult complementary DNA (cDNA), and its sequences showed two isoforms, i.e., the cytosolic and mitochondrial FgTGR. Cytosolic FgTGR (cytFgTGR) was composed of 2370 bp, and its peptide had no signal sequence and hence was not a secreted protein. Mitochondrial FgTGR (mitFgTGR) was composed of 2506 bp with a signal peptide of 43 amino acids; therefore, it was a secreted protein. The putative cytFgTGR and mitFgTGR peptides comprised of 598 and 641 amino acids, respectively, with a molecular weight of 65.8 kDa for cytFgTGR and mitFgTGR, with a conserved sequence (CPYC) of TR, and ACUG and CVNVGC of Grx domains. The recombinant FgTGR (rFgTGR) was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTGR). The FgTGR protein expression, estimated by indirect ELISA using the rabbit anti-rFgTGR as probe, showed high levels of expression in eggs, and 2- and 4-week-old juveniles and adults. The rFgTGR exhibited specific activities in the 5,5'-dithiobis (2-nitro-benzoic acid) (DTNB) reductase assay for TR activity and in β-hydroxyethul disulfide (HED) for Grx activity. When analyzed by immunoblotting and immunohistochemistry, rabbit anti-rFgTGR reacted with natural FgTGR at a molecular weight of 66 kDa from eggs, whole body fraction (WB) of metacercariae, NEJ, 2- and 4-week-old juveniles and adults, and the tegumental antigen (TA) of adult. The FgTGR protein was expressed at high levels in the tegument of 2- and 4-week-old juveniles. The FgTGR 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 a drug target.
authors.affiliation: Department of Pathobiology, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok, 10400, Thailand, narin_bio@hotmail.com.
authors.forename: Narin
authors.initials: N
authors.lastname: Changklungmoa
authors.forename: Pornanan
authors.initials: P
authors.lastname: Kueakhai
authors.forename: Kant
authors.initials: K
authors.lastname: Sangpairoj
authors.forename: Pannigan
authors.initials: P
authors.lastname: Chaichanasak
authors.forename: Wipaphorn
authors.initials: W
authors.lastname: Jaikua
authors.forename: Suda
authors.initials: S
authors.lastname: Riengrojpitak
authors.forename: Prasert
authors.initials: P
authors.lastname: Sobhon
authors.forename: Kulathida
authors.initials: K
authors.lastname: Chaithirayanon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005209:Fasciola / Q000737:chemistry / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D005980:Glutathione Reductase / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport; D011817:Rabbits; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013879:Thioredoxins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D013879:Thioredoxins; D005980:Glutathione Reductase