Cpn0810基因克隆与表达及诱导THP-1细胞产生前炎症细胞因子和凋亡的研究

发布时间：2018-04-19 01:05

本文选题：Cpn0810 + THP-1细胞　；参考：《南华大学》2009年硕士论文

【摘要】：目的:构建重组质粒pGEX6p-2/Cpn0810,在E.coli BL21中进行诱导表达,纯化获得重组融合蛋白Cpn0810。研究该重组蛋白在体外诱导人单核细胞产生前炎症细胞因子TNF-α和IL-6的水平及诱导细胞凋亡的作用,为进一步探索Cpn感染致病的分子机制提供实验依据。方法:PCR扩增Cpn0810蛋白编码基因,构建pGEX6p-2/Cpn0810重组质粒,测序正确后在E.coli BL21中诱导表达,用SDS-PAGE和Western-Blot进行分析和鉴定。用ToxinEraser纯化柱去除内毒素后用不同浓度的GST-Cpn 0810刺激THP-1细胞;ELISA法检测经刺激后的THP-1细胞产生的TNF-α和IL-6水平;以WST-1法检测经GST-Cpn0810处理后对THP-1细胞的增生或抑制作用;用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.coli BL21菌中高效表达出Mr约为42kDa的GST-Cpn 0810目的蛋白,超声裂解后SDS-PAGE分析目的蛋白在菌体细胞内以可溶性形式表达,经采用默克Novagen的GST纯化树脂纯化获得纯度在95%以上的重组蛋白。GST-Cpn 0810能诱导THP-1细胞表达TNF-α和IL-6,并以时间、剂量依赖方式刺激细胞分泌前炎症细胞因子TNF-α和IL-6,当GST- Cpn0810的浓度为4μg/mL时产生的TNF-α和IL-6量最多,分别为(184.75±17.40)pg/mL、(75.36±29.49)pg/mL;当GST-Cpn 0810刺激THP-1细胞24h时产生的TNF-α和IL-6量则达到高峰。另外GST-Cpn 0810以剂量依赖方式抑制THP-1细胞增殖。当10μg/mL GST-Cpn处理THP-1细胞24h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: 1.成功克隆Cpn0810基因并构建了pGEX6p-2/ Cpn0810原核表达载体,将其转化至E.coli BL21后能高效表达出一相对分子质量约42kDa的GST- Cpn0810融合蛋白; 2. Cpn0810蛋白能诱导THP-1细胞表达并分泌前炎症细胞因子TNF-α和IL-6; 3. Cpn 0810蛋白既能抑制THP-1细胞增殖,又能诱导其凋亡。
[Abstract]:Aim: to construct the recombinant plasmid pGEX6p-2 / Cpn0810 and express it in E.coli BL21, and purify the recombinant fusion protein Cpn0810.To study the level of preinflammatory cytokines TNF- 伪 and IL-6 and the role of inducing apoptosis in human monocytes induced by the recombinant protein in vitro, and to provide experimental basis for further exploring the molecular mechanism of Cpn infection.Methods the Cpn0810 protein encoding gene was amplified by 1: -PCR, and the recombinant pGEX6p-2/Cpn0810 plasmid was constructed. The recombinant plasmid was induced and expressed in E.coli BL21 after sequencing correctly, and was analyzed and identified by SDS-PAGE and Western-Blot.The levels of TNF- 伪 and IL-6 produced by stimulated THP-1 cells were detected by Elisa after endotoxin removal by ToxinEraser purification column with different concentrations of GST-Cpn 0810, and the proliferation or inhibition of THP-1 cells was detected by WST-1 method.Apoptosis was detected by Hoechst33258 fluorescence staining and Annexin V-FITC-PI staining.Results: the recombinant plasmid pGEX6p-2/ Cpn0810 was constructed, and the target protein of GST-Cpn 0810, about 42kDa, was highly expressed in E.coli BL21. After ultrasonic lysis, the target protein was expressed in soluble form by SDS-PAGE analysis.Recombinant protein. GST-Cpn 0810 with purity more than 95% was obtained by using GST purified resin of Merck Novagen to induce the expression of TNF- 伪 and IL-6 in THP-1 cells.In a dose-dependent manner, TNF- 伪 and IL-6 were stimulated in a dose-dependent manner. When the concentration of GST- Cpn0810 was 4 渭 g/mL, TNF- 伪 and IL-6 were the highest (184.75 卤17.40 渭 g/mL), 75.36 卤29.49 g / mL, respectively, and when GST-Cpn 0810 stimulated THP-1 cells for 24 h, the production of TNF- 伪 and IL-6 reached its peak.In addition, GST-Cpn 0810 inhibited the proliferation of THP-1 cells in a dose-dependent manner.When THP-1 cells were treated with 10 渭 g/mL GST-Cpn for 24 h, the morphological features of the cells were shrinkage, nuclear fragmentation, cell blister and apoptotic bodies.Conclusion:1.The Cpn0810 gene was cloned successfully and the prokaryotic expression vector of pGEX6p-2/ Cpn0810 was constructed. After being transformed into E.coli BL21, a GST- fusion protein with a relative molecular weight of about 42kDa was expressed efficiently.2.Cpn0810 protein can induce the expression and secretion of proinflammatory cytokines TNF- 伪 and IL-6 in THP-1 cells.3.Cpn 0810 protein can not only inhibit the proliferation of THP-1 cells, but also induce its apoptosis.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

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