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目的:在E.coliBL21中表达肺炎嗜衣原体Cpn0810,并研究该蛋白能否诱导人单核细胞(THP-1)产生TNF-α和IL-6等促炎因子和细胞凋亡。方法:PCR扩增Cpn0810蛋白编码基因,构建pGEX6p-2/Cpn0810重组质粒,在E.coliBL21中诱导表达,经ToxinEraser纯化柱纯化后,用不同浓度的GST-Cpn0810作用THP-1细胞,ELISA法检测TNF-α和IL-6的水平,Hoechst33258荧光染色、AnnexinV-FITC-PI染色法检测细胞凋亡情况。结果:构建了重组质粒pGEX6p-2/Cpn0810,并在E.coliBL21菌中高效表达。该蛋白能诱导THP-1细胞以时间、剂量依赖方式表达TNF-α和IL-6;当10mg/L GST-Cpn0810处理THP-1细胞24 h后,形态学上,细胞表现为皱缩、核碎裂、细胞起泡及凋亡小体等凋亡特征。结论:表达并纯化的Cpn0810蛋白能诱导THP-1细胞分泌TNF-α和IL-6等促炎因子和诱导其发生凋亡。
OBJECTIVE: To express Cpn0810 of Chlamydia pneumoniae in E.coli BL21 and investigate whether this protein can induce proinflammatory cytokine such as TNF-α and IL-6 and apoptosis of human monocytic cells (THP-1). Methods: The gene encoding Cpn0810 protein was amplified by PCR. The recombinant plasmid pGEX6p-2 / Cpn0810 was constructed and expressed in E.coli BL21. After purification by ToxinEraser purification, THP-1 cells were treated with different concentrations of GST-Cpn0810. TNF-α and IL-6, Hoechst33258 staining and Annexin V-FITC-PI staining. Results: The recombinant plasmid pGEX6p-2 / Cpn0810 was constructed and highly expressed in E. coli BL21. The protein could induce THP-1 cells to express TNF-α and IL-6 in a time-and dose-dependent manner. After treatment with 10 mg / L GST-Cpn0810 for 24 h, the cells showed morphological shrinkage and nuclear shrinkage. Fragmentation, cell foaming and apoptotic bodies and other apoptosis characteristics. CONCLUSION: The expressed and purified Cpn0810 protein can induce THP-1 cells to secrete and induce proinflammatory cytokines such as TNF-α and IL-6.