Coloning And Expression Of Cpn0810 Gene From Chlamydophila Pneumoniae And Induce Human Monocytic Cells Express Proinflammatory Cytokines And Apoptosis

Objective:To construct vector pGEX6p-2/Cpn0810 and to expresse the recombinant protein in E.coli BL21.To study the expression and production of proinflamatory cytokines including TNF-αand IL-6 in human monocytic cells (THP-1) and cell apoptosis in human monocytic cells,This will be very useful for the further research of the potential pathogenicity of Chlamydia and its molecular mechanisms.Methods:Polymerase chain reaction(PCR) was used to amplify the Cpn 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 . IPTG was added to induce the expression of the recombinant protein .The expressed protein was identitied by SDS-PAGE and Western blot. 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-αand IL-6 by ELISA . Inhibition of cell proliferation on THP-1 cells treated with Cpn0810 was assessed by WST-1. 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.The similarity had 100% between the inserted gene with Cpn0810 gene reported in Genbank by BLAST analysis.The SDS-PAGE demonstrated that the GST-Cpn0810 recombinant protein with relative molecular weight about 42kDa was expressed after the E.coli BL21 containing the recombinant plasmid was induced by IPTG, and mainly existed in the pattern of solubility body. Its purity reached up to 95% after purification with GST resin chromatography of Novagen, Cpn0810 stimulated THP-1 cell to produce proinflamatory cytokines including TNF-αand IL-6 in a dose and time-dependent manner. There was an optimal concentration of Cpn0810 at 4μg/mL in the induction of TNF-αand IL-6 (184.75±17.40pg/mL,75.36±29.49pg/mL, respectively). The induced TNF-αand IL-6 production reached peak levels at 24h of stimulation with Cpn0810. Otherwise, Cpn0810 inhibited the growth of THP-1 cell in a dose-dependent manner. After THP-1 cells were treated with 10μg/mL Cpn0810 for 24h, apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy; apoptosis of cell was detected after 24h in THP-1 cells treated with Cpn0810.Conclusion:1. pGEX6p-2/Cpn0810 prokaryotic expression vector was successfully constructed,and then transformed into E.coli BL21. A recombinant protein with relative molecular weight near 42kDa was expressed;2. Cpn0810 recombinant protein could stimulate THP-1 cell to produce and express proinflamatory cytokines including TNF-αand IL-6;3. Cpn0810 recombinant protein not only inhibite the proliferation but also induce the apoptosis of THP-1 in vitro.