The Effect Of Chlamydia Psittaci MIP On The Production Of Proinflammatory Cytokines And Apoptosis In Host Cells

Objective: To study the ability of Chlamydia psittaci Microphage infectivity potentiator(MIP)inducing proinflammatory cytokines production and apotosis inhibition of host cells.This will be very useful for the further research of the molecular pathogenic mechanisms of C.psittaci.Methods: PCR was used to amplify the MIP gene fragment from C.psittaci 6BC chromosomal DNA and the MIP gene fragment was cloned into p ET-30a(+)vector and the prokaryotic expression vector of p ET-30(+)/MIP was constructed.The recombinant protein was expressed in E.coli BL21 with IPTG induction and identitied by SDS-PAGE.After used polymyxin B to remove endotoxin,different concentrations of the recombinant MIP protein were used to stimulate THP-1 cells to produce IL-8 and TNF-? with various durations.After treated with C.psittaci MIP,the apoptosis of He La cells was tested with fluorescence staining and flow cytometry.Results: A fragment of the target gene was amplified with PCR.p ET-30(+)/MIP prokaryotic expression vector was successfully constructed.After PCR,restriction enzymes cleavage analysis and sequencing,it was proved that the target gene was inserted.The DNA sequence of the cloned MIP gene was the same as that published on Gen Bank.The SDS-PAGE demonstrated that the recombinant protein with relative molecular weight about 34 k Da was expressed in E.coli BL21 induced by IPTG.Its purity reached up to 95% after purification with HIS resin chromatography.C.psittaci MIP stimulated THP-1 cells to produce proinflamatory cytokines including IL-8 and TNF-? in a dose and time-dependent manner.When the concentration of C.psittaci MIP was 16 ?g/m L,the production of IL-8 and TNF-? reached the highest level(105.01 pg/m L,50.52 pg/m L,respectively).The release of IL-8 and TNF-? was increasing over time.The results of fluorescent hoechst32258 staining and Flow cytometric assay showed that the number of apoptosis cells in C.psittaci MIP treated cells was significantly reduced.Conclusion: 1.p ET-30(+)/MIP prokaryotic expression vector was successfully constructed,and a recombinant protein with relative molecular weight about 34 k Da was expressed in E.coli BL21.2.Recombinant C.psittaci MIP protein could stimulate THP-1 cells to produce proinflamatory cytokines including IL-8 and TNF-?.3.Recombinant C.psittaci MIP could inhibit He La cells apoptosis.