[Construction, expression and kinase function analysis of an eukaryocyte vector of rhoptry protein 17 in Toxoplasma gondii].

OBJECTIVE

To construct and express the eukaryocytic expression vector of rhoptry protein 17 of Toxoplasma gondii RH strain (TgROP17) and analyze its kinase function.

METHODS

The open reading frame of TgROP17 gene was amplified from total RNA in T. gondii RH strain by RT-PCR, and cloned into p3 x Flag-CMV-14 vector to construct recombinant plasmid p3xFlag-CMV-14/TgROP17. After colony-PCR confirming, double restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector p3xFlag-CMV-14/TgROP17 was transfected into HEK 293T cells. The target gene was examined by RT-PCR and the recombinant protein was detected by Western blotting. The kinase activity of TgROP17 was identified by Western blotting and its apoptotic function was assessed by flow cytometry.

RESULTS

The size of RT-PCR product was 1 850 bp. The recombinant plasmid p3xFlag-CMV-14/ TgROP17 was confirmed by colony-PCR, double restriction enzyme digestion and DNA sequencing. RT-PCR and Western blotting analysis showed that TgROP17 was expressed in the p3xFlag-CMV-14/TgROPl7 transfected-HEK 293T cells rather than in mock cells. The amplified gene was with 1,850 bp and the target protein was about M, 70,000. Western blotting analysis showed that c-Jun was phosphorylated by TgROP17. Flow cytometry analysis indicated that camptothecin-induced apoptosis was inhibited by TgROP17 with an inhibition rate of 20.6% and 24.1% at 6 h and 12 h after coculture, respectively, which was higher than that of the control (P < 0.05).

CONCLUSION

The eukaryotic expression vector p3xFlag-CMV-14/TgROP17 is constructed. TgROP17 has kinase activity and playes an anti-apoptosis role.