Chlamydial protease-like activity factor from Chlamydophila pneumoniae induced THP-1 cells produced proinflammtory cytokines and apoptosis / 中华微生物学和免疫学杂志

ABSTRACT

Objective To express and purify Chlamydial protease-like activity factor(CPAF)from Chlamydophila pneumoniae,for investigating the effect of its recombinant protein GST-CPAF in inducing human monocytic cells to secrete proinflammatory cytokines and cell apoptosis.Methods The recom-bination expression plasmid pGEX6p-2/CPAF from Chlamydophila pneumoniae was transformed into E.coli.The recombination GST-CPAF was expressed after induction by IPTG,and purified by a agarose gel FF.Human monocytic cells were stimulated by the GST-CPAF to test the production of tumor necrosis factor a(TNF-α)and interleukin-6(IL- 6)by ELISA.Inhibition of cells proliferation with GST-CPAF was assessed by MTT.The THP-1 cell apoptosis stimulated by GST-CPAF was detected by Hoechst33258 fluorescence staining,DNA fragmentation analysis and cell apeptosis was detested bv Annexin V-FITC-propidiuum iodide (PI)staining.Results The recombination protein GST-CPAF was successfully expressed with high level in E.coli,and stimulated human monocytic cells to produce proinflammatory cytokines including TNF-α and IL-6 in a dose-and time-dependent manner.Otherwise,the GST-CPAF inhibited the growth of human monocytic cell in a dose-dependent manner.Apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy,DNA ladders in apoptosis cells were detected after 24 h with the GST-CPAF.Conclusion The GST-CPAF from Chlamydophila pneumoniae can induce the secretion of proinflammatory cytokines TNF-α and IL-6 by human monocytic cells,and inhibited the proliferation of THP-1 cell and apoptosis in vitro.