Abstract
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.
Highlights
Toxoplasma gondii is a protozoan parasite that infects all mammals and birds and causes toxoplasmosis
An alignment of the amino acid sequence of granule protein (GRA)-4 from the local isolate which was cloned with granule protein 4 (GRA-4) was obtained from NCBI database and showed 99.61% homology to the predicted GRA-4 from the T. gondii Izatnagar isolate
After conducting Basic Local Alignment Search Tool (BLAST) runs for the predicted amino acid sequence using sequence deposited in GenBank, the sequence of GRA-4 gene from the local isolate showed homology with several T. gondii GRA-4 genes as presented in Table-1
Summary
Toxoplasma gondii is a protozoan parasite that infects all mammals and birds and causes toxoplasmosis. The prevalence rate of T. gondii infection varies between countries, with infection rates between 10% and 80% of the population [1]. Animals consumed by humans are often infected with T. gondii muscle cysts, a source of parasite transmission to humans, which remain a public health concern [2]. Toxoplasmosis diagnosis consists of either a serological test using monoclonal antibody (fluorescent antibody, ELISA), polymerase chain reaction (PCR), molecular probes (DNA tracking), or dipstick test, in conjunction with clinical symptoms. Toxoplasma antigens have been detected in several domestic cat organs using immunohistochemistry with antigen labeled-(strept) Avidin-Biotin (LAB-SA) [3]. Hanafiah et al [4] detected 33.3% toxoplasmosis prevalence in cats using
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