title: [Induction of proinflammatory cytokines and cell apoptosis by Chlamydia pneumoniae Cpn0810].
abstract: OBJECTIVE
  To expresse the Chlamydia pneumoniae Cpn0810 in E.coli BL21, and to study weather could it inducing proinflamatory cytokines including TNF-α and IL-6 in human monocytic (THP-1) and cell apoptosis.
  
  
  METHODS
  Polymerase chain reaction(PCR) was used to amplify the Cpn0810 gene, PCR products were purified and cloned into the prokaryotic expression vector pGEX6p-2. The restriction plasmids pGEX6p-2/Cpn0810 confirmed by PCR and sequencing was transformed into E.coli BL21. The recombinant protein was purified with glutathione S-transferase (GST) resin chromatography of Novagen after renaturation. THP-1 cells were stimulated by different concentrations of Cpn0810 and for various durations to test the production and the expression of TNF-α and IL-6 by ELISA. Cell apoptosis was detected in C.pneumoniae Cpn0810 cells by Hoechst33258 fluorescence staining and Cell apoptosis was detected in THP-1 cells by Annexin-V-FITC-propidiu-m iodide (PI) staining.
  
  
  RESULTS
  The restriction enzymes cleavage analysis and nucleotide sequencing showed the target gene was successfully inserted into pGEX6p-2 prokaryotic expression vector. Cpn0810 stimulated THP-1 cell to produce proinflamatory cytokines including TNF-α and IL-6 in a dose and time-dependent manner. After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy; apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810.
  
  
  CONCLUSIONS
  Cpn0810 recombinant protein could stimulate THP-1 cell to produce and express proinflamatory cytokines including TNF-α and IL-6; After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810.
authors: Department of Pathogenic Biology, University of South China, Hengyang 421001, China.
authors: Yu-Yu
authors: YY
authors: Chen
authors: Yi-Mou
authors: YM
authors: Wu
authors: Liang-Zhuan
authors: LZ
authors: Liu
authors: Li-Li
authors: LL
authors: Chen
authors: Qiu-Gui
authors: QG
authors: Zhang
mesh_terms: D017209:Apoptosis / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology*; D002460:Cell Line; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000276:immunology*; D016207:Cytokines / Q000096:biosynthesis*; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D016207:Cytokines; D018836:Inflammation Mediators; D015850:Interleukin-6; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha