| Objective:In order to provide experimental basis for exploring pathogenic mechanism of Chlamydophila pneumoniae (Cpn) ,the recombinant expression vector containing the 181～400aa region gene(CPAFm) of the Chlamydial protease-like activity factor(CPAF)from Cpn was induced and expressed in Escherichia coli (E.coli) and the recombinant GST-CPAFm protein was purified to investigate the changes of pulmonary inflammation and inflammatory cytokines including TNF-αand IL-6 levels in BALB/c mouse.Methods:The recombinant plasmid pGEX6p-2/CPAFm was transformed into E.coli BL21 after being identified by PCR, enzymes cleavage analysis and sequencing and then the E.coli BL21 containing the recombinant plasmid was induced by IPTG, the recombinant protein was expressed and analyzed by SDS-PAGE and Western blot. The recombinant protein was purified with (glutathione S-transferase, GST) agarose gel FF after renaturation and its concentration were determined by A280 ultraviolet spectrophotometry. The purified recombinant protein was instilled into anterior nose or injected into the caudal vein of BALB/c mouse on day 1, 3 and 6. On day 7 BALB/c mouse were killed. Pathological alteration was observed by hematoxylin and eosin staining, the total white blood cells of peripheral blood and bronchoalveolar lavage fluid (BALF) was counted with blood cell counting plate, the leukocyte differential count in peripheral blood and BALF was observed by Wright's taining, the concentration of TNF-αand IL-6 in plasma and BALF was detected by Enzyme-linked immunoadsordent assay technique.Results:1) The pGEX6p-2/CPAFm recombinant plasmid expressed a relative molecular weight about 51.3kDa protein in the E.coli BL21 through IPTG induction ,and mainly existed in the pattern of inclusion body. Its purity reached up to 95% after purification with GST agarose gel FF. The Western blot proved that it could specifically react with mouse anti-GST monoclonal antibodies.2) Histological examination of BALB/c mouse lung tissue after intranasal or intravenous injection application of 100μg GST-CPAFm showed a marked inflammation immune responsonse and bronchiole and perivascular leukocyte infiltration such as neutrophils,lymphocytes and macrophages ,and the control and normal group BALB/c mice lung tissue no obvious pathological changes.3) The percentage of peripheral blood neutrophils in GST-CPAFm intravenously group significantly higher than that in the GST group (P﹤0.01), PBS group (P﹤0.01) and normal group (P﹤0.01), and mononuclear cells was significantly higher than that in GST group (P﹤0.05), PBS group (P﹤0.05) and normal group (P﹤0.01).4) The number of total leukocytes of BALF in GST-CPAFm intranasally group was significantly higher than that in the GST group (P﹤0.05), PBS group (P﹤0.05) and normal group (P﹤0.01).5) The level of inflammatory cytokines IL-6 of the peripheral blood in GST-CPAFm intranasally group were significantly higher than that in the GST group (P﹤0.01), PBS group (P﹤0.01) and normal group (P﹤0.01), the level of inflammatory cytokines TNF-αwere significantly higher than the GST group (P﹤0.05), PBS group (P﹤0.01) and normal group (P﹤0.01); and the level of inflammatory cytokines IL-6 in GST-CPAFm intravenously group were significantly higher than that in the GST group (P﹤0.01), PBS group (P﹤0.01) and normal group (P﹤0.01). 6) The level of inflammatory cytokines IL-6 and TNF-αof BALF in GST-CPAFm intranasally group were significantly higher than that in the GST,PBS and normal group (P﹤0.01) ; and the level of inflammatory cytokines IL-6 and TNF-αof BALF in GST-CPAFm intravenously group were significantly higher than the GST,PBS and normal group (P﹤0.01) .Conclusion1. About 51.3 kDa GST-CPAFm recombinant protein and 26 kDa GST protein were successful expressed and purified;2. GST-CPAFm recombinant protein could induce inflammatory injury of the lung tissue in BALB/c mouse;3. GST-CPAFm recombinant protein could induce the increase of inflammatory cells and the expression of inflammatory cytokines including TNF-α, IL-6 in BALF of BALB/c mouse.
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