[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.