Dataset Information

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

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 TA^R 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.

SUBMITTER: Hanafiah M

PROVIDER: S-EPMC7704308 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Cloning and expression of <i>Toxoplasma gondii</i> GRA-4 recombinant protein as a toxoplasmosis diagnostic kit candidate.

Hanafiah Muhammad M Helmi Teuku Zahrial TZ Sutriana Amalia A Priyowidodo Dwi D Fihiruddin Fihiruddin F

Veterinary world 20201005 10

<h4>Aim</h4>The objective of this study was to produce recombinant protein GRA-4 (rGRA-4) of a local <i>Toxoplasma gondii</i> isolate as a candidate for a toxoplasmosis diagnosis kit in <i>Escherichia coli</i> BL21 (DE3) competent cells using pET SUMO plasmid.<h4>Materials and methods</h4>Samples used were stock <i>T. gondii</i> tachyzoites DNA from the Parasitology Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta. Amplified GRA-4 polymerase chain reaction product o ...[more]

PMID: 33281340

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Project description:Toxoplasma gondii is a ubiquitous protozoan intracellular parasite, the causative agent of toxoplasmosis, and a worldwide zoonosis for which an effective vaccine is needed. Actin is a highly conserved microfilament protein that plays an important role in the invasion of host cells by T. gondii. This study investigated the immune responses elicited by BALB/c mice after nasal immunisation with a recombinant T. gondii actin (rTgACT) and the subsequent protection against chronic and lethal T. gondii infections. We evaluated the systemic response by proliferation, cytokine and antibody measurements, and we assessed the mucosal response by examining the levels of TgACT-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes. Parasite load was assessed in the liver and brain, and the survival of mice challenged with a virulent strain was determined. The results showed that the mice immunised with rTgACT developed high levels of specific anti-rTgACT IgG titres and a mixed IgG1/IgG2a response with a predominance of IgG2a. The systemic immune response was associated with increased production of Th1 (IFN-γ and IL-2), Th2 (IL-4) and Treg (IL-10) cytokines, indicating that not only Th1-type response was induced, but also Th2- and Treg-types responses were induced, and the splenocyte stimulation index (SI) was increased in the mice immunised with rTgACT. Nasal immunisation with rTgACT led to strong mucosal immune responses, as seen by the increased secretion of SIgA in nasal, vaginal and intestinal washes. The vaccinated mice displayed significant protection against lethal infection with the virulent RH strain (survival increased by 50%), while the mice chronically infected with RH exhibited lower liver and brain parasite loads (60.05% and 49.75%, respectively) than the controls. Our data demonstrate, for the first time, that actin triggers a strong systemic and mucosal response against T. gondii. Therefore, actin may be a promising vaccine candidate against toxoplasmosis.

Dataset Information

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

Publications

Cloning and expression of <i>Toxoplasma gondii</i> GRA-4 recombinant protein as a toxoplasmosis diagnostic kit candidate.

Similar Datasets

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