Prokaryotic Expression And Peroxidases Enzyme Activity Of Ahpc Protein From C. Trachomatis

Objective: The aim of this study was to investigate the production of ROS in macrophage cells which were infected with Chlamydia trachomatis and identify the antioxidant activity of Ct Ahpc protein. Methods: The macrophage cells were infected with Ct and the cells were collected at 0h, 3h, 6h, 9h and 12 h. Production of reactive oxygen species(ROS) was detected by fluorescent probe and the m RNA levels of Ahpc were quantified by Realtime-PCR during the different infection time. The Ahpc gene was amplified by polymerase chain reaction(PCR) from Ct genome DNA using a pair of specific primers, then Ahpc gene was subcloned into vector p ET28 a to construct prokaryotic expression vector p ET28a-Ahpc. The p ET28a-Ahpc was transformed into E.coli BL21 and recombinant Ahpc protein was induced by IPTG. The recombinant Ahpc protein was purified through the Ni2+-affinity chromatography. The enzyme activity of Ahpc was detected by ferrous oxidation xylenol orange(FOX) assay. The growth profiles of E.colis which harboring the p ET28a-Ahpc or p ET28 a in the presence of paraquat were detected. Results:(1) Ct induced the production of ROS in macrophage cells, the ROS reached the peak level at 6h after infection. The mean fluorescent intensities of ROS is 1980±310 at 3h, 4260±350 at 6h, 2900±245 at 12 h and 2100±250 at 24 h, respectively.(2) The realtime PCR showed the relative m RNA expression of Ahpc was significantly increased at 3, 6, 9 or 12 h with 7.3, 8.2, 5.2 or 4.5 fold, respectively during infection(3) The Ahpc gene with 588 bp in length was amplified from the Ct genome and was subcloned into a p ET28 a expression vector. The recombinant vector p ET28a-Ahpc was constructed and transformed into E.coli BL21 sucessfully.(4) The Ahpc protein with the moulecular weight of 26 k Da was induced by IPTG and was purified successfully by Ni2+-affinity chromatography.(5) The t-BHP and H2O2 were degraded in vitro by Ahpc protein.(6) The E. coli transformed with p ET28a-Ahpc showed significantly improved tolerance to oxidative stress which caused by paraquat compared to E.coli transformed with an empty p ET28 a vector. Conclusions: C. trachomatis induced an increase in the ROS level and the m RNA expression of Ahpc was upregulated during infection. The Ahpc protein confered resistance to oxidative stress in vitro and in E.coli.