The Study On Antioxidant Mechanism Of Toxoplasma Gondii Prx In Vitro

Objective:To prokaryotic express the peroxiredoxin(Prx)of Toxoplasma gondii RH strain,and produce the polyclonal antibody. To express Prx in Hep G2 cells with the constructed eukaryotic expression vector. Using different final concentration of H2O2 to induce the host cell apoptosis, then detecting the changes of intracellular ROS generation, to analyze the role of Prx and the mechanism of Prx antioxidant.Methods:To construct the prokaryotic expression vector Prx/p ET-28a(+), then being efficiently expressed in E. coil Rosetta by changing ITPG concentration, and induction time and temperature. The anti-Prx polyclone antibody was prepared by the purified recombinant protein in rabbits.The specificity of polyclonal antibody was detected by the Western blotting.After construct the eukaryotic expression vector p3XFLAG-Myc-CMVTM-24-Prx, the recombinant plasmid was extracted by plasmid Maxi Kit(endotoxin-free) and transfected into Hep G2 cells by Ronfect TM. In 24 h,48 h,72 h after transfection, Hep G2 cells were collected, and the expression of Prx was determined by Western Blotting with the polyclonal antibody.Hep G2 cells were cultured in 24-well plates for 24 h, then transfected by p3XFLAG-Myc-CMVTM-24(empty plasmid group) and p3XFLAG-Myc-CMVTM-24-Prx(transfection group), and added the final concentration of 200 μmol/L 、 400μmol/L 、800 μmol/L and 1 600μmol/L H2O2. After transfection 48 h, the cells were collected and stained by Annexin V-FITC/PI, then the cell apoptosis rates were detected by flow cytometry. Meanwhile, the generation of ROS in cells were detected by flow cytometry with DCFH-DA staining.Results:Western blotting could detect the specificity of the anti-Prx polyclonal antibody,which will facilitate the study of the biological functions of Prx.After transfection 24 h, 48 h, 72 h, the special band of 25 KDa, which was equal size with Prx, could be detected by Western blotting respectively, and the protein expression at 48 h was the highest.At 48 h after transfection, in final concentration of 200 μmol/L、400μmol/L and800μmol/L H2O2, the apoptosis rates of transfection group were lower than that of empty plasmid group, and the apoptosis rates of the two groups had significant difference. In 1 600μmol/L H2O2,there were no significant difference between the two groups.At 48 h after transfection, in final concentration of 200 μmol/L、400μmol/L and800μmol/L H2O2, the ROS generation of transfection was lower than that of empty plasmid group, and the differences between the two groups were statistical significant.In 1 600μmol/L H2O2, there were no significant difference between the two groups.Conclusions:Anti-Prx polyclonal antibody has good specificity.Prx was expressed successfully in Hep G2 cells, and Prx has the effect of antioxidant by reducing the levels of the generation of ROS in cells.