{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,19]],"date-time":"2026-02-19T08:01:58Z","timestamp":1771488118076,"version":"3.50.1"},"reference-count":24,"publisher":"Veterinary World","issue":"10","license":[{"start":{"date-parts":[[2020,10,5]],"date-time":"2020-10-05T00:00:00Z","timestamp":1601856000000},"content-version":"vor","delay-in-days":278,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2020,10,5]],"date-time":"2020-10-05T00:00:00Z","timestamp":1601856000000},"content-version":"am","delay-in-days":278,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2020,10,5]],"date-time":"2020-10-05T00:00:00Z","timestamp":1601856000000},"content-version":"tdm","delay-in-days":278,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100009509","name":"Kementerian Riset Teknologi Dan Pendidikan Tinggi Republik Indonesia","doi-asserted-by":"publisher","award":["019\/UN11.2\/LT\/SP3\/2019"],"award-info":[{"award-number":["019\/UN11.2\/LT\/SP3\/2019"]}],"id":[{"id":"10.13039\/501100009509","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vet World"],"published-print":{"date-parts":[[2020]]},"abstract":"<jats:p>Aim: The objective of this study was to produce recombinant protein GRA-4 (rGRA-4) of a local Toxoplasma gondii isolate as a candidate for a toxoplasmosis diagnosis kit in Escherichia coli BL21 (DE3) competent cells using pET SUMO plasmid.\n\nMaterials and Methods: Samples used were stock T. gondii tachyzoites DNA from the Parasitology Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta. Amplified GRA-4 polymerase chain reaction product of T. gondii tachyzoite DNA was cloned in the pET-SUMO TAR cloning vector. The GRA-4 gene from T. gondii local isolate was sequenced, followed by plasmid transformation, recombinant plasmid DNA isolation, and recombinant protein expression in DE3 competent cells.\n\nResults: The amplification product of GRA-4 T. gondii gene was 1036 bp, with 48 kDa molecular weight after expression in DE3 competent cells. An alignment of the amino acid sequence of GRA-4 from the local isolate which was cloned with GRA-4 was obtained from NCBI database and showed 99.61% homology to the predicted GRA-4 from the T. gondii Izatnagar isolate. Amino acid sequence of the predicted GRA-4 protein from local isolate was different at positions 19 and 304.\n\nConclusion: This research cloned rGRA-4 in pET SUMO plasmid.<\/jats:p>","DOI":"10.14202\/vetworld.2020.2085-2091","type":"journal-article","created":{"date-parts":[[2020,10,5]],"date-time":"2020-10-05T08:09:57Z","timestamp":1601885397000},"page":"2085-2091","source":"Crossref","is-referenced-by-count":5,"title":["Cloning and expression of Toxoplasma gondii GRA-4 recombinant protein as a toxoplasmosis diagnostic kit candidate"],"prefix":"10.14202","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1491-8766","authenticated-orcid":false,"given":"Muhammad","family":"Hanafiah","sequence":"first","affiliation":[{"name":"Parasitology Laboratory, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8623-2854","authenticated-orcid":false,"given":"Teuku Zahrial","family":"Helmi","sequence":"additional","affiliation":[{"name":"Laboratory of Biochemistry, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6669-7491","authenticated-orcid":false,"given":"Amalia","family":"Sutriana","sequence":"additional","affiliation":[{"name":"Pharmacology Laboratory, Faculty of Veterinary Medicine, Universitas Syiah Kuala Banda Aceh, Indonesia."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5528-8035","authenticated-orcid":false,"given":"Dwi","family":"Priyowidodo","sequence":"additional","affiliation":[{"name":"Department of Parasitology, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta, Indonesia."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8803-0463","authenticated-orcid":false,"given":"Fihiruddin","family":"Fihiruddin","sequence":"additional","affiliation":[{"name":"Department of Medical Laboratory Technology, Politeknik Kemenkes Mataram, Sandubaya Mataram Nusa Tenggara Barat Indonesia."}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"5308","published-online":{"date-parts":[[2020,10,5]]},"reference":[{"key":"ref0","doi-asserted-by":"publisher","unstructured":"Robert-Gangneux, F. and Darde, M.L. (2012) Epidemiology of and diagnostic strategies for toxoplasmosis. Clin. Microbiol. Rev., 25(2): 264-296.","DOI":"10.1128\/cmr.05013-11"},{"key":"ref1","doi-asserted-by":"publisher","unstructured":"Zhou, D.H., Zhao, F.R., Lu, P., Xia, H.Y., Xu, M.J., Yuan, L.G., Yan, C., Huang, S.Y., Li, S.J. and Zhu, X.Q. (2012) Seroprevalence of Toxoplasma gondii infection in dairy cattle in southern China. Parasit. Vectors, 5(1): 48-52.","DOI":"10.1186\/1756-3305-5-48"},{"key":"ref2","doi-asserted-by":"publisher","unstructured":"Hanafiah, M., Nurcahyo, R.W., Siregar, R.Y., Prastowo, J., Hartati, S., Sutrisno, S. and Aliza, D. (2017) Detection of Toxoplasma gondii in cat\u2019s internal organs by immunohistochemistry methods labeled with-[strept] avidin-biotin. Vet. World, 10(9): 1035-1039.","DOI":"10.14202\/vetworld.2017.1035-1039"},{"key":"ref3","doi-asserted-by":"publisher","unstructured":"Hanafiah, M., Prastowo, P., Hartati, S., Aliza, D. and Nurcahyo, R.W. (2018) Detection of Toxoplasma gondii copro-prevalence by polymerase chain reaction using repetitive 529 bp gene in feces of pet cats (Felis catus) in Yogyakarta, Indonesia. Vet. World, 11(9): 338-1343.","DOI":"10.14202\/vetworld.2018.1338-1343"},{"key":"ref4","unstructured":"Suwanti, L. T. (2015) The role of veterinarian in toxoplasmosis control through development of diagnostic kit and immunotherapy. Universitas Airlangga Surabaya. Printed by Airlangga University Press. pp.1-34."},{"key":"ref5","doi-asserted-by":"publisher","unstructured":"Meng, M., Zhou, A., Lu, G., Wang, L., Zhao, G., Han, Y., Zhou, H., Cong, H., Zhao, Q., Zhu, XQ. and He, S. (2013) DNA prime and peptide boost immunization protocol encoding the Toxoplasma gondii GRA-4 induces strong protective immunity in BALB\/c mice. BMC Infect. Dis., 13(1): 494-504.","DOI":"10.1186\/1471-2334-13-494"},{"key":"ref6","unstructured":"Golkar, M., Shokrgozar, M.A., Rafati, S., Sodaie, M.R. and Assmar, M. (2005) Construction, expression and preliminary immunological evaluation of a DNA plasmid encoding the GRA2 protein of Toxoplasma gondii. Iran. Biomed. J., 9(1): 1-8."},{"key":"ref7","doi-asserted-by":"publisher","unstructured":"Cuppari, A.F., Sanchez, V., Ledesma, B., Frank, F.M., Goldman, A., Angel, S.O. and Martin, V. (2008) Toxoplasma gondii protease inhibitor-1 (TgPI-1) is a novel vaccine candidate against toxoplasmosis. Vaccine, 26(39): 5040-5045.","DOI":"10.1016\/j.vaccine.2008.07.031"},{"key":"ref8","doi-asserted-by":"publisher","unstructured":"S\u00e1nchez, V.R., Pitkowski, M.N. and Cuppari, F.A.V. (2011) Combination of CPG-oligodeoxynucleotides with recombinant ROP2 or GRA4 proteins induces protective immunity against Toxoplasma gondii infection. Exp. Parasitol., 128(4): 448-453.","DOI":"10.1016\/j.exppara.2011.04.004"},{"key":"ref9","doi-asserted-by":"publisher","unstructured":"S\u00e1nchez, V.R., Fenoy, I.M., Picchio, M.S., Soto, A.S., Nadia, A., Goldman, A. and Martin, V. (2015) Homologous prime-boost strategy with TgPI-1 improves the immune response and protects highly susceptible mice against chronic Toxoplasma gondii infection. Acta Trop., 150: 159-165.","DOI":"10.1016\/j.actatropica.2015.07.013"},{"key":"ref10","unstructured":"Invitrogen. (2002) BL21 StarTM (DE3) One Shot\u00ae. Available from: https:\/\/assets.thermofisher.com\/TFS-Assets\/LSG\/manuals\/oneshot_mach1_man.pdf. Retrieved on 23-09-2020."},{"key":"ref11","unstructured":"Hanafiah, M., Helmi, T.Z., Amalia, Fihiruddin, and Bahi, M. (2020) Nucleotide plasmid and DNA Recombinant Sequence GRA-4 Toxoplasma gondii. The 2nd International Conference on Veterinary, Animal, and Environmental Science (ICVAES 2020). FKH. Darussalam Banda Aceh. (In press)."},{"key":"ref12","unstructured":"Invitrogen (2010) ChampionTM pET SUMO Protein Expression System, Manual part no. 25-0709. Life Technology. Available from: https:\/\/assets.thermofisher.com\/TFS-Assets\/LSG\/manuals\/petsumo_man.pdf. Retrieved on 25-09-2020."},{"key":"ref13","doi-asserted-by":"publisher","unstructured":"Garcia- R\u00e9quet, N., Lebrun, M., Fourmaux, M., Mercereau-Puijalon, O., Taramann, Beckers, J.M., Samyn, B., Van Beeumen, J., Bout, D. and Dubremetz, J. (2000) The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite. Cell. Microbiol., 2(4): 353-364.","DOI":"10.1046\/j.1462-5822.2000.00064.x"},{"key":"ref14","unstructured":"Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbour Laboratory Press, New York."},{"key":"ref15","doi-asserted-by":"publisher","unstructured":"Dewi, N.N.D. (2006) Gene Cloning of the Micronema Protein 3 (MIC3) Local Isolate of Tachyzoite Toxoplasma gondii. Thesis. Biotechnology Studies Program Universitas Gadjah Mada, Yogyakarta.","DOI":"10.22146\/ijbiotech.7410"},{"key":"ref16","doi-asserted-by":"crossref","unstructured":"McPherson, M.J. and Moller, S.G. (2006) PCR. 2nd ed. Taylor and Francis Group, Madison Avenue, New York. p270.","DOI":"10.4324\/9780203002674"},{"key":"ref17","unstructured":"Marjanh, A., Ghaffarifar, F., Forooghi, F., Dalimi, A.H., Sharifi, Z. and Maspi, N. (2018) Expression of plasmid encoded GRA-4 Gene of Toxoplasma gondii RH strain in CHO eukaryotic cells. Iran. J. Parasitol., 13(3): 392-398."},{"key":"ref18","unstructured":"Indrasanti, D., Haryanto, A. and Artama, W.T. (2011) Subcloning and isolation of the micronema gene 3 (MIC-3) local isolate of Toxoplasma gondii. Molecule, 6(1): 10-18."},{"key":"ref19","unstructured":"Glick, B.R. and Pasternak, J.J. (1994), Molecular Biotechnology. Principles and Applications of Recombinant DNA. ASM Press, Washington DC. p17-33."},{"key":"ref20","unstructured":"Brown, T.A. (2010) Gene Cloning: An Introduction. Sixth.Edition. London: Chapman and Hall. p13-43."},{"key":"ref21","unstructured":"Primrose, S.B., Twyman, R. and Old, R.W. (2002), Principles of Gene Manipulation. 6th ed. Wiley-Blackwell. p145-170."},{"key":"ref22","doi-asserted-by":"publisher","unstructured":"Ram, H., Rao, J.R., Tewari, A.K., Banerjee, P.S. and Sharma, A.K. (2013) Molecular cloning, sequencing, and biological characterization of GRA-4 gene of Toxoplasma gondii. Parasitol. Res., 112(7): 2487-2494.","DOI":"10.1007\/s00436-013-3414-3"},{"key":"ref23","doi-asserted-by":"publisher","unstructured":"Martin, V., Supanitsky, A. and Echeverria, P.C. (2004) Recombinant GRA-4 or ROP2 protein combined with alum or the GRA-4 gene provides partial protection in chronic murine models of toxoplasmosis. Clin. Diagn. Lab. Immunol., 1(4): 704-710.","DOI":"10.1128\/cdli.11.4.704-710.2004"}],"container-title":["Veterinary World"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/www.veterinaryworld.org\/Vol.13\/October-2020\/6.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"http:\/\/www.veterinaryworld.org\/Vol.13\/October-2020\/6.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"text-mining"},{"URL":"http:\/\/www.veterinaryworld.org\/Vol.13\/October-2020\/6.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2020,11,30]],"date-time":"2020-11-30T12:05:49Z","timestamp":1606737949000},"score":1,"resource":{"primary":{"URL":"http:\/\/www.veterinaryworld.org\/Vol.13\/October-2020\/6.html"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020]]},"references-count":24,"issue-title":["October-2020"],"journal-issue":{"issue":"10","published-online":{"date-parts":[[2020,10,1]]},"published-print":{"date-parts":[[2020]]}},"URL":"https:\/\/doi.org\/10.14202\/vetworld.2020.2085-2091","relation":{},"ISSN":["2231-0916","0972-8988"],"issn-type":[{"value":"2231-0916","type":"electronic"},{"value":"0972-8988","type":"print"}],"subject":[],"published":{"date-parts":[[2020]]}}}