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

Aim: The objective of this study was to produce recombinant protein GRA-4 (rGRA-4) of a local isolate as a candidate for a toxoplasmosis diagnosis kit in BL21 (DE3) competent cells using pET SUMO plasmid.

Materials And Methods: Samples used were stock tachyzoites DNA from the Parasitology Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta. Amplified GRA-4 polymerase chain reaction product of tachyzoite DNA was cloned in the pET-SUMO TA cloning vector. The gene from 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 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 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.