Coloning And Expression Of Cpn0425 Recombinant Protein From Chlamydophila Pneumoniae And Induce Human Monocytic Cells Express Proinflammatory Cytokines And Apoptosis

Objective:The recombinant expression vector of the Cpn0425 from Chlamydophila pneumoniae was constructed and expressed in E.coli.It,s induced effect on expression of proinflammatory secreted by monocytes and on apoptois. After purified, the recombinant protein (Cpn0425) was to investigate the expression and production of proinflamatory cytokines including IL-8 and IL-1βin human monocytic cells (THP-1);zhe and apoptosis in human monocytic cells,This study was designed to investigated the potential pathogenicity of Chlamydia and its molecular mechanisms responsible for the induction of proinflammatory cytokines and apoptosis.Methods:The plasmid transformed into E.coli BL21 after which was construeted and identified, and then analyzed by SDS-PAGE and Western blot.It was purified with GST(glutathione S-transferase, GST) resin chromatography of Novagen after renaturation and its concentrations were determined by A280 ultraviolet spectrophotometry. THP-1 cells were stimulated by different concentrations of C. pneumoniae Cpn0425 and the expression of IL-8 and IL-1βwere tested and proirferation by ELISA . Zhe inhibition of cell treated with Cpn0425 was assessed by WST-1. Cell apoptosis was detected by Hoechst33258 ,DNA fragmentation analysis and Annexin-V-FITC- PI. 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 Cpn0425 gene reported in Genbank by BLAST analysis.The SDS-PAGE demonstrated that the GST-Cpn0425 recombinant protein with relative molecular weight about 50kDa was expressed after the E.coliBlLue containing the recombinant plasmid was induced , and mainly existed in the pattern of solubility body.Its purity reached up to 95% after purification with GST(glutathione S-transferase, GST) resin chromatography of Novagen, C. pneumoniae Cpn0425 stimulated THP-1 cell to express the mRNA of IL-8 and IL-1βand produce proinflamatory cytokines including IL-8 and IL-1βin a dose and time-dependent manner. There was an optimal concentration of C. pneumoniae Cpn0425 at 6μg/mL in the induction of IL-8 (716.11±41.26pg/mL) and there was an optimal concentration of C. pneumoniae Cpn0425at 8μg/mL in the induction of IL-1β(32.91±5.49pg/mL). The induced IL-8 and IL-1βproduction reached peak levels at 24h of stimulation with Cpn0425. Otherwise, Cpn0425 inhibited the growth of THP-1 cell in a dose-dependent manner. After THP-1 cells were treated with 12μg/mL C. pneumoniae Cpn0425 for 48h, apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent microscopy.Conclusion:1. pGEX6p-2/Cpn0425 prokaryotic expression vector was successfully constructed,and then transformed into E.coli. A recombinant protein with relative molecular weight about 50kDa was expressed;2. Cpn0425 recombinant protein could stimulate THP-1 cell to produce and express the proinflamatory cytokines which were including IL-8 and IL-1β;3. Cpn0425 recombinant protein could not only inhibite the proliferation but also induce the apoptosis of THP-1.