Browsing by Author "Tepparit Samrit"

Filter results by typing the first few letters
Now showing 1 - 4 of 4
  • No Thumbnail Available
    PublicationMetadata only
    Expression and characterization of glutathione peroxidase of the liver fluke, Fasciola gigantica
    (2018-11-01) Narin Changklungmoa; Kulathida Chaithirayanon; Werachon Cheukamud; Athit Chaiwichien; Supawadee Osotprasit; Tepparit Samrit; Prasert Sobhon; Pornanan Kueakhai; Mahidol University; Burapha University
    © 2018, Springer-Verlag GmbH Germany, part of Springer Nature. Glutathione peroxidase (GPx) is a key member of the family of antioxidant enzymes in trematode parasites including Fasciola spp. Because of its abundance and central role as an anti-oxidant that helps to protect parasites from damage by free radicals released from the host immune cells, it has both diagnostic as well as vaccine potential against fasciolosis. In this study, we have cloned, characterized, and detected the expression of the GPx protein in Fasciola gigantica (Fg). FgGPx (582 bp) was cloned by polymerase chain reaction (PCR) from complementary DNA (cDNA) from an adult fluke. Its putative peptide has no signal sequence and is composed of 168 amino acids, with a molecular weight of 19.1 kDa, and conserved sequences at NVACKUG, FPCNQFGGQ, and WNF. Phylogenetic analysis showed that GPx is present from protozoa to mammals and FgGPx was closely related to Fasciola hepatica GPx. A recombinant FgGPx (rFgGPx) was expressed in Escherichia coli BL21 (DE3) and used for immunizing mice to obtain polyclonal antibodies (anti-rFgGPx) for immunoblotting and immunolocalization. In immunoblotting analysis, the FgGPx was expressed in all stages of F. gigantica (eggs, metacercariae, newly excysted juveniles (NEJ), 4-week-old juveniles, and adults). This mouse anti-rFgGPx reacted with the native FgGPx at a molecular weight of 19.1 kDa in adult whole body (WB) and tegumental antigens (TA) as detected by immunoblotting. The FgGPx protein was expressed at a high level in the tegument, vitelline glands, and eggs of the parasite. Anti-rFgGPx exhibited no cross-reactivity with the other parasite antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. The possibility of using rFgGPx for immunodiagnosis and/or as a vaccine for fasciolosis in animals of economic importance will be explored in the future.
  • No Thumbnail Available
    PublicationMetadata only
    Monoclonal antibody against Fasciola gigantica glutathione peroxidase and their immunodiagnosis potential for fasciolosis
    (2019-12-01) Pornanan Kueakhai; Kulathida Chaithirayanon; Athit Chaiwichien; Tepparit Samrit; Supawadee Osotprasit; Phawiya Suksomboon; Wipaphorn Jaikua; Prasert Sobhon; Narin Changklungmoa; Mahidol University; Burapha University
    © 2019 Elsevier B.V. Glutathione peroxidases (GPx), major antioxidant enzymes, secreted by Fasciola spp., are important for the parasite evasion and protection against the host's immune responses. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica glutathione peroxidase (rFgGPx) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgGPx. This MoAb (named 7B8) is IgG1 with κ light chains, and it reacted specifically with rFgGPx at a molecular weight 19 kDa as shown by immunoblotting, and reacted with the native FgGPx in the extracts of whole body (WB), metacercariae, newly excysted juveniles (NEJs), 4 week-old juveniles and adult F. gigantica as shown by indirect ELISA. It did not cross react with antigens in WB fractions from other adult trematodes, including Fischoederius cobboldi, Paramphistomum cervi, Setaria labiato-papillosa, Eurytrema pancreaticum, Gastrothylax crumenifer and Gigantocotyle explanatum. By immunolocalization, MoAb against rFgGPx reacted with the native protein in the tegument, vitelline cells, and eggs of adult F. gigantica. In addition, the sera from mice experimentally infected with F. gigantica were tested positive by this indirect sandwich ELISA. This result indicated that FgGPx is an abundantly expressed parasite protein that is secreted into the tegumental antigens (TA), therefore, FgGPx and its MoAb may be used for immunodiagnosis of both early and late fasciolosis gigantica in animals and humans.
  • No Thumbnail Available
    PublicationMetadata only
    A novel Thioredoxin-related protein 14 from Fasciola gigantica has an immunodiagnostic potential for fasciolosis
    (2020-07-01) Narin Changklungmoa; Pornanan Kueakhai; Kant Sangpairoj; Supawadee Osotprasit; Athit Chaiwichien; Tepparit Samrit; Prasert Sobhon; Kulathida Chaithirayanon; Mahidol University; Thammasat University; Burapha University
    © 2020 Elsevier B.V. In the definitive host, a trematode parasite can survive and evade the damage by reactive oxygen species that are generated from its metabolism and the host immune cells. Several anti-oxidant proteins are found in Fasciola spp. which play essential roles in cellular redox balance. One of them is thioredoxin-related protein 14 (TRP14) that has a highly conserved WCPDC motif and serves as a disulfide reductase-like thioredoxin (Trx). In the present study, a cDNA encoding TRP14 from F. gigantica (FgTRP14) was selected and cloned by immunoscreening with a rabbit infected serum. Phylogenetic analysis was performed by MEGA X program showed that FgTRP14 was most highly related to the Fasciola hepatica. Immunoblotting analysis of the polyclonal antibody rabbit serum against recombinant FgTRP14 (rFgTRP14) revealed that the molecular weight of natural FgTRP14 was at 14 kDa from metacercariae, NEJ, 4-week old juvenile and adult stage. The native FgTRP14 was expressed in caecal epithelial cells and preferentially localized on the cells’ surface lamellae of adult stage. By sandwich ELISA assay, the circulating FgTRP14 could be recognized in sera of experimentally F. gigantica metacercariae infection in mice. The native FgTRP14 in the excretory–secretory (ES) and whole body (WB) of adult F. gigantica were detected at the concentrations 6.3 ng/ml, and 45 ng/ml, respectively. Therefore, it could be considered for immunodiagnostic candidate for fasciolosis.
  • No Thumbnail Available
    PublicationMetadata only
    Toxicity and Anti-Oxidation Capacity of the Extracts from Caulerpa Lentillifera
    (2021-07-01) Supawadee Osotprasit; Tepparit Samrit; Athit Chaiwichien; Narin Changklungmoa; Krai Meemon; Nakorn Niamnont; Preeyanuch Manohong; Kunwadee Noonong; Montakan Tamtin; Prasert Sobhon; Pornanan Kueakhai; Walailak University; Mahidol University; Burapha University; King Mongkut's University of Technology Thonburi; Coastal Fisheries Research and Development Bureau
    Caulerpa lentillifera (sea grape) has been widely used in pharmaceutical industry and health-care products in Thailand. In this study, we attempted to evaluate the toxicity and antioxidant capacity of sea grape extracts in five fractions (ethanol- CLET, hexane- CLHE, ethyl acetate- CLEA, butanol-CLBU, and aqueous-CLAQ). The extracts were evaluated for cytotoxicity by MTT and LDH assays on four cell lines, fibroblast (L929), macrophages (RAW 264.7), hepatocytes (FL83B), and keratinocytes (HaCaT). Genotoxicity was tested by comet assay and micronucleus assay on human lymphoblast cells (TK6). The antioxidant capacity was measured by DPPH and ABTS scavenging assays. Our results demonstrated low cytotoxicity and genotoxicity of CLET, CLBU and CLAQ. When tested by DPPH and ABTS assays, CLET, CLEA, and CLHE showed high antioxidant activity. In conclusion, CLET, CLBU, and CLAQ demonstrated no toxic effects, and CLET, CLEA, and CLHE exhibited high antioxidant capacity. Therefore, our results indicated that CLET, CLEA, and CLHE could be consumed safely at doses lower than 500 and 200 μg/ml for CLHE and CLEA, respectively.