Cloning And Expression Of Chlamydia Pneumoniae Heat Shock Protein 10 Gene And Study Of Its Induction Of Proinflammatory Cytokines And Apoptosis

Objective:In order to provide experimental basis for exploring pathogenic molecular machanism of Chlamydial pneumoniae (C. pneumoniae), C.pneumoniae heat shock protein 10 (Hsp10) gene was amplified by PCR technique to construct recombinant plasmid pQE30/Hsp10, and recombinant HsplO fusion protein (rHsp10) was expressed and purified to investigate the expression and production of proinflamatory cytokines including TNF-α and IL-6 in mouse macrophages; cell apoptosis in mouse macrophages stimulated by C.pneumoniae Hsp10 was also detected.Methods:A pair of primers of C.pneumoniae HsplO gene were designed and the C. pneumoniae Hsp10 gene was amplified from C. pneumoniae AR39 complete genome by PCR.The gene was directly ligated into pUCm-T vector .After identifying by PCR , restriction enzyme analysis and sequence analysis, the gene of Hsp10 was subcloned into the expression vector pQE30 to generate recombinant plasmid pQE30/Hsp10. The fusion protein was expressed in E. coli M15 and purified with Ni-NTA affinity chromatography. Murine macrophages line RAW264.7 were stimulated by different concentrations of rHsp10 and for various durations to test the production and the expression of TNF-α and IL-6 by ELIS A and RT-PCR. Inhibition of cell proliferation on RAW264.7 treated with rHsp10 was assessed by MTT assay. Cell apoptosis was detected in RAW264.7 cells by Hoechst33258 fluorescence staining DNA fragmentation analysis and Annexin-FITC Apoptosis Detection Kit.