关键词:  , 刚地弓形虫, 棒状体蛋白17, 真核表达, 丝氨酸?鄄苏氨酸激酶, 细胞凋亡

Abstract: 【Abstract】 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×Flag-CMV-14 vector to construct recombinant plasmid p3×Flag-CMV-14/TgROP17. After colony-PCR confirming, double restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector p3×Flag-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 p3×Flag-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 p3×Flag-CMV-14/TgROP17 transfected-HEK 293T cells rather than in mock cells. The amplified gene was with 1 850 bp and the target protein was about Mr 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 co-culture, respectively, which was higher than that of the control(P＜0.05).  Conclusion  The eukaryotic expression vector p3×Flag-CMV-14/TgROP17 is constructed. TgROP17 has kinase activity and playes an anti-apoptosis role.

Key words:  , Toxoplasma gondii；Rhoptry protein 17；Eukaryotic expression；Serine-threonine kinase；Apoptosis