Cloning and expression of Toxoplasma gondii GRA-4 recombinant protein as a toxoplasmosis diagnostic kit candidate

Vet World Vol.13 October-2020 Article-6

Research Article

Veterinary World, 13(10): 2085-2091

https://doi.org/10.14202/vetworld.2020.2085-2091

Muhammad Hanafiah¹, Teuku Zahrial Helmi², Amalia Sutriana³, Dwi Priyowidodo⁴, and Fihiruddin Fihiruddin⁵

1. Parasitology Laboratory, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia.
2. Laboratory of Biochemistry, Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh, Indonesia.
3. Pharmacology Laboratory, Faculty of Veterinary Medicine, Universitas Syiah Kuala Banda Aceh, Indonesia.
4. Department of Parasitology, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta, Indonesia.
5. Department of Medical Laboratory Technology, Politeknik Kemenkes Mataram, Sandubaya Mataram Nusa Tenggara Barat Indonesia.

Background and 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.

Materials 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.

Results: 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.

Conclusion: This research cloned rGRA-4 in pET SUMO plasmid. Keywords: cloning, expression, GRA-4, pET-SUMO, plasmid, recombinant.

Keywords: cloning, expression, GRA-4, pET-SUMO, plasmid, recombinant.

How to cite this article: Hanafiah M, Helmi TZ, Sutriana A, Priyowidodo D, Fihiruddin F (2020) Cloning and expression of Toxoplasma gondii GRA-4 recombinant protein as a toxoplasmosis diagnostic kit candidate, Veterinary World, 13(10): 2085-2091.

Received: 31-03-2020 Accepted: 24-08-2020 Published online: 05-10-2020

Corresponding author: Muhammad Hanafiah E-mail: hanafi_2015@unsyiah.ac.id

DOI: 10.14202/vetworld.2020.2085-2091

Copyright: Hanafiah, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.