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.