Chen Yu-Yu, Wu Yi-Mou, Liu Liang-Zhuan, Chen Li-Li, Zhang Qiu-Gui
Department of Pathogenic Biology, University of South China, Hengyang 421001, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2011 Sep;27(9):954-8.
To expresse the Chlamydia pneumoniae Cpn0810 in E.coli BL21, and to study weather could it inducing proinflamatory cytokines including TNF-α and IL-6 in human monocytic (THP-1) and cell apoptosis.
Polymerase chain reaction(PCR) was used to amplify the Cpn0810 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. 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-α and IL-6 by ELISA. 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.
The restriction enzymes cleavage analysis and nucleotide sequencing showed the target gene was successfully inserted into pGEX6p-2 prokaryotic expression vector. Cpn0810 stimulated THP-1 cell to produce proinflamatory cytokines including TNF-α and IL-6 in a dose and time-dependent manner. After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis with nuclear chromatin fragmentation as well as cell shrinkage was observed by fluorescent staining and microscopy; apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810.
Cpn0810 recombinant protein could stimulate THP-1 cell to produce and express proinflamatory cytokines including TNF-α and IL-6; After THP-1 cells were treated with 10 mg/L Cpn0810 for 24 h, apoptosis of cell was detected after 24 h in THP-1 cells treated with Cpn0810.
在大肠杆菌BL21中表达肺炎衣原体Cpn0810，并研究其是否能诱导人单核细胞（THP-1）产生包括肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）在内的促炎细胞因子以及细胞凋亡。
采用聚合酶链反应（PCR）扩增Cpn0810基因，对PCR产物进行纯化并克隆至原核表达载体pGEX6p-2。经PCR和测序确认的重组质粒pGEX6p-2/Cpn0810转化至大肠杆菌BL21。复性后，用诺维信的谷胱甘肽S-转移酶（GST）树脂层析法纯化重组蛋白。用不同浓度的Cpn0810刺激THP-1细胞不同时长，通过酶联免疫吸附测定（ELISA）检测TNF-α和IL-6的产生及表达。用Hoechst33258荧光染色检测肺炎衣原体Cpn0810细胞中的细胞凋亡，用膜联蛋白-V-异硫氰酸荧光素-碘化丙啶（PI）染色检测THP-1细胞中的细胞凋亡。
限制性内切酶切割分析和核苷酸测序表明目的基因已成功插入pGEX6p-2原核表达载体。Cpn0810以剂量和时间依赖性方式刺激THP-1细胞产生包括TNF-α和IL-6在内的促炎细胞因子。用10mg/L Cpn0810处理THP-1细胞24小时后，通过荧光染色和显微镜观察到细胞核染色质碎片化以及细胞收缩的凋亡现象；在用Cpn0810处理的THP-1细胞中，24小时后检测到细胞凋亡。
Cpn0810重组蛋白可刺激THP-1细胞产生并表达包括TNF-α和IL-6在内的促炎细胞因子；用10mg/L Cpn0810处理THP-1细胞24小时后，在用Cpn0810处理的THP-1细胞中，24小时后检测到细胞凋亡。
