Cloning and Expression of Recombinant Glutathione Peroxidase in Fasciola Gigantica

Abstract

Glutathione peroxidase (GPx) is one of the most essential antioxidant enzymes in trematode parasites that lacks catalase. GPx has been already characterized in the trematodes such as Schistoma mansoni, Clonorchis sinensis, and Paragonimus westermani. However, there is no reported about GPx in Fasciola spp. This study amplified GPx cDNA of Fasciola gigantica (FgGPx) from cDNA library. Its sequence contained an in-frame TGA codon for selenocysteine (Sec) which is a stop codon in prokaryote. The Sec (TGA) codon was mutated into a sense codon for cysteine (TGC) using site-directed mutagenesis. The recombinant modified FgGPx (rM_FgGPx) protein was expressed as intracellular protein in Escherichia coli. Polyclonal anti-rM_FgGPx was produced and immunoblotting analysis demonstrated that the anti-rM_FgGPx antibodies did not cross-react with crude antigens from other trematodes including S. mansoni, Opisthorchis viverrini and Paramphistomum spp. The result of ELISA showed that test group had a significant increase of total immunoglobulin and IgG2a when compared with control group. Moreover, there was a significant increase of total immunoglobulin in several F. gigantica infected animal including mice, rabbits and cows, but no cross reaction between O. viverrini infected hamsters sera and rM_FgGPx. Therefore, rM_FgGPx could be used as an immunodiagnostic tool for fascioliasis.