Charoonluk Jirapattharasate1, Ruenruetai Udonsom2, Apichai Prachasuphap3, Kodcharad Jongpitisub3, Panadda Dhepakson3. 1. Department of Preclinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, 999 Phutthamonthon sai 4 Rd, Salaya, Nakhonpathom, 73170, Thailand. Charoonluk.jir@mahidol.edu. 2. Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Ratchathewi, Bangkok, 10400, Thailand. 3. Department of Medical Sciences, Medical Life Sciences Institute, 88/7 Tiwanon Road, Talad Kwan Subdistrict, Muang District, Nonthaburi, 11000, Thailand.
Abstract
BACKGROUND: The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT). RESULTS: Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT. CONCLUSION: Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.
BACKGROUND: The development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT). RESULTS: Western blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT. CONCLUSION: Our findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.
Authors: Ragab M Fereig; Hassan Y A H Mahmoud; Samy G A Mohamed; Mahmoud Rezk AbouLaila; Azza Abdel-Wahab; Salama Ahmed Osman; Sherif Abdallah Zidan; Sabry A El-Khodary; Adel Elsayed Ahmed Mohamed; Yoshifumi Nishikawa Journal: Vet Parasitol Reg Stud Reports Date: 2016-05-11
Authors: S Stelzer; W Basso; J Benavides Silván; L M Ortega-Mora; P Maksimov; J Gethmann; F J Conraths; G Schares Journal: Food Waterborne Parasitol Date: 2019-04-03