淋球菌PorB重组蛋白诱导THP-1分泌促炎细胞因子和细胞凋亡的研究
发布时间：2019-03-01 07:59
【摘要】：目的：构建pGEX-6p-1/porB原核重组质粒，表达并纯化GST-PorB融合蛋白，分析重组PorB蛋白诱导人单核细胞白血病细胞（THP-1）分泌促炎细胞因子IL-1β、TNF-α及HMGB1的能力，并探讨PorB蛋白对诱导THP-1细胞凋亡作用的影响。 方法：从Genbank获得PorB蛋白的全基因序列，设计引物，使用PCR技术扩增出PorB蛋白的基因序列，通过基因克隆技术将该段序列插入到pGEX-6p-1中，构建pGEX-6p-1/porB原核表达载体，以双酶切及测序鉴定载体构建是否成功。将构建好的载体转入到制备好的Ecoli-BL-21感受态细菌中，诱导表达PorB蛋白，摸索最适诱导表达条件；通过尿素浓度梯度复性法对包涵体PorB蛋白进行复性，使用GST-Bind树脂纯化复性后的PorB蛋白。BCA法测定纯化后的蛋白浓度，使用多粘菌素-B去除蛋白中的内毒素，鲎试剂盒测定内毒素含量。PorB蛋白经过处理后分别以时间梯度和浓度梯度作用于THP-1细胞， ELISA双抗体夹心法分别检测IL-1β、TNF-α和HMGB1的产生水平；采用流式细胞术和AnnexinV-EGFP-PI双染色法观察不同时间和不同浓度的PorB蛋白对诱导THP-1细胞凋亡作用的影响。 结果： (1)正确克隆porB基因，并将porB基因插入到pGEX-6p-1载体中，成功构建了porB/pGEX-6p-1原核表达载体。 (2)摸索出重组蛋白的最适表达条件，即在30℃时，以0.2mM IPTG诱导表达菌表达4h时，蛋白表达效果最好。经纯化后的重组蛋白，通过SDS-PAGE电泳，只出现大小为59KD左右的单一条带，符合GST-PorB融合蛋白的预测值。 (3)分别以浓度为1，3，5，，7，9μg/ml的重组porB蛋白处理THP-1细胞24h，在蛋白浓度为5μg/ml时，TNF-α，IL-1β和HMGB1分泌量达到最高峰，分别为（27.10±1.23）pg/ml，（109.44±3.53）pg/ml，（320.09±9.67）pg/ml，与PBS/GST对照组及其它各浓度组比较具有显著差异(P0.05)。使用5μg/ml浓度的重组蛋白分别刺激THP-1细胞6，12，24，36，48h，TNF-α，IL-1β在刺激时间为36h时分泌量最高，分别为（39.94±2.35）pg/ml，（223.14±7.27）pg/ml，与其它各时间点组比较具有显著差异(P0.05)，而HMGB1分泌量从6h至48小时之间一直呈上升趋势。 (4)分别以浓度为1，3，5，7，9μg/ml的重组porB蛋白浓度处理THP-1细胞24h，通过AnnexinV-EGFP-PI荧光双染法和流式细胞术检测细胞凋亡情况，在蛋白浓度为7μg/ml时，细胞凋亡情况最明显，凋亡百分率为（23.13±2.01）％，与PBS/GST对照组及其它各浓度组比较具有显著差异(P0.05)；使用7μg/ml的蛋白处理细胞0，12，24，36，48h，结果显示在24h时细胞凋亡率达到最高，为（20.75±2.03）％，与其它各时间点组比较具有显著差异(P0.05)。 结论： (1) PorB重组蛋白能通过时间和剂量依赖方式诱导THP-1细胞分泌较高水平的促炎细胞因子IL-1β、TNF-α和HMGB1。 (2) PorB重组蛋白能通过时间和剂量依赖方式诱导THP-1细胞发生凋亡。
[Abstract]:Objective: to construct the prokaryotic recombinant plasmid of pGEX-6p-1/porB, express and purify GST-PorB fusion protein, and analyze the ability of recombinant PorB protein to induce the secretion of pro-inflammatory cytokines IL-1 尾, TNF- 伪 and HMGB1 by human monocytic leukemia cells (THP-1). The effect of PorB protein on apoptosis of THP-1 cells was also investigated. Methods: the whole gene sequence of PorB protein was obtained from Genbank, the primers were designed, the gene sequence of PorB protein was amplified by PCR, and inserted into pGEX-6p-1 by gene cloning technology. The prokaryotic expression vector of pGEX-6p-1/porB was constructed, and the success of the vector was identified by double enzyme digestion and sequencing. The constructed vector was transferred into the prepared Ecoli-BL-21 competent bacteria to induce the expression of PorB protein and to explore the optimal conditions for inducing the expression. The inclusion body PorB protein was renatured by urea concentration gradient renaturation, the renatured PorB protein was purified by GST-Bind resin, the purified protein concentration was determined by BCA method, and the endotoxin in the protein was removed by polymyxin-B. THP-1 cells were treated with time gradient and concentration gradient, respectively. IL-1 尾, TNF- 伪 and HMGB1 production levels were detected by ELISA double antibody sandwich method. The effects of different time and concentration of PorB protein on apoptosis of THP-1 cells were observed by flow cytometry and AnnexinV-EGFP-PI double staining. Results: (1) the porB gene was cloned correctly, and the porB gene was inserted into the pGEX-6p-1 vector. The prokaryotic expression vector of porB/pGEX-6p-1 was constructed successfully. (2) the optimal expression condition of the recombinant protein was found, that is, at 30 鈩
本文编号：2432239
[Abstract]:Objective: to construct the prokaryotic recombinant plasmid of pGEX-6p-1/porB, express and purify GST-PorB fusion protein, and analyze the ability of recombinant PorB protein to induce the secretion of pro-inflammatory cytokines IL-1 尾, TNF- 伪 and HMGB1 by human monocytic leukemia cells (THP-1). The effect of PorB protein on apoptosis of THP-1 cells was also investigated. Methods: the whole gene sequence of PorB protein was obtained from Genbank, the primers were designed, the gene sequence of PorB protein was amplified by PCR, and inserted into pGEX-6p-1 by gene cloning technology. The prokaryotic expression vector of pGEX-6p-1/porB was constructed, and the success of the vector was identified by double enzyme digestion and sequencing. The constructed vector was transferred into the prepared Ecoli-BL-21 competent bacteria to induce the expression of PorB protein and to explore the optimal conditions for inducing the expression. The inclusion body PorB protein was renatured by urea concentration gradient renaturation, the renatured PorB protein was purified by GST-Bind resin, the purified protein concentration was determined by BCA method, and the endotoxin in the protein was removed by polymyxin-B. THP-1 cells were treated with time gradient and concentration gradient, respectively. IL-1 尾, TNF- 伪 and HMGB1 production levels were detected by ELISA double antibody sandwich method. The effects of different time and concentration of PorB protein on apoptosis of THP-1 cells were observed by flow cytometry and AnnexinV-EGFP-PI double staining. Results: (1) the porB gene was cloned correctly, and the porB gene was inserted into the pGEX-6p-1 vector. The prokaryotic expression vector of porB/pGEX-6p-1 was constructed successfully. (2) the optimal expression condition of the recombinant protein was found, that is, at 30 鈩
本文编号：2432239
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