Molecular cloning, sequencing, and biological characterization of GRA4 gene of Toxoplasma gondii

In the present study, GRA4 (dense granule antigen) gene of Toxoplasma gondii was cloned, sequenced, and biologically characterized. The nucleotide sequence data obtained were analyzed and submitted in GenBank database (accession no. EU660037). Analysis of nucleotide sequence of GRA4 gene revealed 99.2 % homology with the published sequence (accession no. M76432). The gene segment (open reading frame) of 1,054 bp was further amplified and re-cloned in expression vector pET-32a. The recombinant protein obtained following the expression in prokaryotic system had a molecular mass of approx. 50 kDa and showed good immunoreactivity with T. gondii sera collected from infected goats. The immunization study of the recombinant protein performed in laboratory mice and live challenge with T. gondii revealed a high level of IgG response against the tachyzoite lysate antigen (TLA) by an indirect ELISA. Protection against T. gondii challenge infection was not evident in immunized mice except for the prolongation of survival period by 2 days. Humoral immune response profile revealed initially a high level of IgG antibody, but at 1 week post-challenge, a sudden drop in the level of the antibody was appreciable. Cytokine profiling by enzyme-linked immunosorbent spot method revealed relatively high level of IFN-γ production by the rodent spleen cells followed by IL-10 and IL-4. Increase in IFN-γ production by spleen cells of immunized mice following TLA stimulation suggested direct correlation to the up-regulated Th1 cells. However, the present immunization trial failed to show any positive relationship with the protection of mice following T. gondii challenge infection.