Publication: Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase
Issued Date
2015-02-24
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ISSN
14321955
09320113
09320113
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2-s2.0-84939997449
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Mahidol University
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SCOPUS
Bibliographic Citation
Parasitology Research. Vol.114, No.6 (2015), 2119-2127
Suggested Citation
Narin Changklungmoa, Pornanan Kueakhai, Kant Sangpairoj, Pannigan Chaichanasak, Wipaphorn Jaikua, Suda Riengrojpitak, Prasert Sobhon, Kulathida Chaithirayanon Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase. Parasitology Research. Vol.114, No.6 (2015), 2119-2127. doi:10.1007/s00436-015-4400-8 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/35200
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Title
Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase
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Abstract
© 2015, Springer-Verlag Berlin Heidelberg. 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.