梅毒螺旋体膜脂蛋白Tp0663、Tp0821、Tp0971通过TLR2途径诱导巨噬细胞产生前炎症细胞因子
发布时间：2019-03-10 21:33
【摘要】： 目的:探讨梅毒螺旋体(Treponema pallidum, Tp)膜脂蛋白Tp0663、Tp0821、Tp0971能否诱导巨噬细胞产生前炎症细胞因子(CKs)IL-6和IL-1β;通过观察TLR2抗体、CD14抗体、NF-κB特异抑制剂二硫代氨基甲酸吡咯烷(PDTC)对三种膜脂蛋白诱导巨噬细胞产生CKs的影响,研究这些膜脂蛋白诱导产生前炎症CKs是否与TLR2、CD14及NF-κB介导的信号转导途径有关。 方法:①通过Genbank获取选Tp0663、Tp0821和Tp0971的基因序列,以Tp Nichols株基因组DNA为模板,PCR扩增目的片段,将其亚克隆至原核表达载体pET28a(+)中构建重组质粒pET28a(+)/Tp0663、pET28a(+)/Tp0821和pET28a(+)/Tp0971,经PCR、双酶切、测序鉴定后将其转化至表达宿主菌E.coli Rosseta中,IPTG诱导表达。采用SDS-PAGE和Western blot分析和鉴定表达产物;Ni-NTA亲和层析柱纯化重组蛋白,BCA法测定蛋白浓度;Detoxi-Gel?内毒素去除胶去除重组蛋白中的内毒素,经鲎试剂测定内毒素含量。②佛波酯(PMA)诱导人单核细胞THP-1转化为巨噬细胞,用Tp0663、Tp0821和Tp0971重组蛋白刺激巨噬细胞,ELISA双抗体夹心法检测诱生其前炎症细胞因子IL-6和IL-1β的情况;用TLR2抗体、CD14抗体和PDTC预处理巨噬细胞后,用Tp0663、Tp0821和p0971重组蛋白分别刺激巨噬细胞,ELISA分析TLR2抗体、CD14抗体和PDTC对重组蛋白诱导巨噬细胞产生IL-6和IL-1β的影响。 结果:构建的重组质粒经酶切和测序鉴定证实插入片段为目的基因,测序结果与Genbank上登录序列完全一致;SDS-PAGE结果显示,在IPTG诱导下,重组表达菌分别表达了一相对分子量(Mr)约为34kDa(Tp0663)和36 kDa(Tp0821),29 kDa(Tp0971)的重组蛋白,目的蛋白在菌体细胞内以可溶性和包涵体形式存在,经Ni-NTA亲和纯化后,纯度在95%以上,Western blot结果显示其与目的蛋白大小相符;内毒素去除胶处理重组蛋白,经鲎试剂检测内毒素小于0.04EU/mL;THP-1细胞经PMA刺激转化为巨噬细胞,不同浓度的重组蛋白(0.5μg/ml～10μg/ml)能明显诱导巨噬细胞产生IL-6和IL-1β;当重组蛋白浓度高于3μg/ml(Tp0663)和5μg/ml(Tp0821和Tp0971)时,IL-6和IL-1β产生的量无明显增加。TLR2抗体处理后,Tp0663, Tp0821和Tp0971诱导IL-6的产生量分别降至66.0%、64.0%和60.0%,IL-1β的产生量分别降至63.0%、62.0%和62.0%;经CD14抗体处理后, IL-6的产生量分别降至71.0%、73.0%和69.0%,IL-1β的产生量分别降至69.0%、70.0%和66.0%,经TLR2和CD14抗体联合处理后IL-6的产生量分别降至54.0%、55.0%和56.0%,IL-1β的产生量分别降至51.0%、50.0%和52.0%,NF-κB特异性抑制剂PDTC处理后IL-6和IL-1β的产生量分别降至20%以下,各处理组与对照组(同型抗体)比较(P0.05)有明显差异。 结论: 1. Tp0663、Tp0821和Tp0971重组蛋白能诱导巨噬细胞产生前炎症CKs IL-6和IL-1β; 2. Tp0663、Tp0821和Tp0971重组蛋白诱导巨噬细胞产生前炎症CKs与TLR2和CD14及NF-κB参与的信号转导通路有关。
[Abstract]:Objective: to investigate whether Treponema pallidum (Treponema pallidum, Tp) membrane lipoprotein Tp0663,Tp0821,Tp0971 can induce the production of proinflammatory cytokines (CKs) IL-6 and IL-1 尾 by macrophages. By observing the effects of TLR2 antibody, CD14 antibody and NF- kappa B specific inhibitor pyrrolidine dithiocarbamate (PDTC) on the production of CKs in macrophages induced by three membrane lipoproteins, the effects of these membrane lipoproteins on the pre-production of CKs and TLR2, were studied. The signal transduction pathway mediated by CD14 and NF- 魏 B is related. Methods: 1 the gene sequences of selected Tp0663,Tp0821 and Tp0971 were obtained by Genbank, and the target fragment was amplified by PCR using genomic DNA of Tp Nichols strain as template. The recombinant plasmid pET28a () / Tp0663, was subcloned into prokaryotic expression vector pET28a () to construct the recombinant plasmid pET28a () / Tp0663,. PET28a () / Tp0821 and pET28a () / Tp0971, were digested by PCR, and identified by sequencing. They were transformed into the host strain E.coli Rosseta and induced by IPTG. The expression products were analyzed and identified by SDS-PAGE and Western blot, the recombinant protein was purified by Ni-NTA affinity chromatography, the protein concentration was determined by BCA, and the protein concentration was determined by Detoxi-Gel?. Endotoxin was removed from the recombinant protein and the endotoxin content was measured by Limulus lysate. 2 (PMA) induced the transformation of human monocytes THP-1 into macrophages, and the macrophages were stimulated by Tp0663,Tp0821 and Tp0971 recombinant proteins. The proinflammatory cytokines IL-6 and IL-1 尾 were detected by ELISA double antibody sandwich method. After macrophages were pretreated with TLR2 antibody, CD14 antibody and PDTC, the macrophages were stimulated with Tp0663,Tp0821 and p0971 recombinant protein, respectively. The effects of TLR2 antibody, CD14 antibody and PDTC on IL-6 and IL-1 尾 induced by recombinant protein were analyzed by ELISA. Results: the recombinant plasmid was confirmed to be the target gene by restriction endonuclease digestion and sequencing. The result of sequencing was identical with the login sequence of Genbank. The results of SDS-PAGE showed that under the induction of IPTG, a recombinant protein with relative molecular weight of 34kDa (Tp0663), 36 kDa (Tp0821) and 29 kDa (Tp0971) was expressed respectively. The target protein existed in the form of soluble and inclusion bodies in the cell. After purification with Ni-NTA affinity, the purity of the protein was over 95%. The results showed that it was consistent with the size of the target protein. The recombinant protein was removed by lipopolysaccharide (LPS), and the endotoxin was less than 0.04 EU / mL by Limulus lysate (Limulus lysate). THP-1 cells were transformed into macrophages by PMA stimulation. Different concentrations of recombinant protein (0.5 渭 g / ml~ 10 渭 g / ml) could significantly induce the production of IL-6 and IL-1 尾 by macrophages. When the concentration of recombinant protein was higher than 3 渭 g / ml (Tp0663) and 5 渭 g / ml (Tp0821 and Tp0971), the production of IL-6 and IL-1 尾 did not increase significantly. The production of IL-6 induced by Tp0663, Tp0821 and Tp0971 decreased to 66.0% after treatment with TLR 2 antibody. The production of IL-1 尾 decreased to 63.0%, 62.0% and 62.0%, respectively, at 64.0% and 60.0%, respectively. After treatment with CD14 antibody, the production of IL-6 decreased to 71.0%, 73.0% and 69.0%, and the production of IL-1 尾 decreased to 69.0%, 70.0% and 66.0%, respectively, and the production of IL-1 尾 decreased to 71.0%, 73.0% and 69.0%, respectively. The production of IL-6 and IL-1 尾 decreased to 54.0%, 55.0% and 56.0%, respectively, and IL-1 尾 production decreased to 51.0%, 50.0% and 52.0% after combined treatment of TLR2 and CD14 antibody, respectively, and the production of IL-1 尾 decreased to 51.0%, 50.0% and 52.0%, respectively. The production of IL-6 and IL-1 尾 decreased to less than 20% after treatment with NF- kappa B specific inhibitor PDTC, and there was significant difference between each treatment group and the control group (P0.05). Conclusions: 1. The recombinant proteins of Tp0663,Tp0821 and Tp0971 can induce the production of CKs IL-6 and IL-1 尾 in macrophages. The pre-inflammatory CKs induced by Tp0663,Tp0821 and Tp0971 recombinant proteins is related to the signal transduction pathways involved in TLR2, CD14 and NF- 魏 B.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R377
本文编号：2438050
[Abstract]:Objective: to investigate whether Treponema pallidum (Treponema pallidum, Tp) membrane lipoprotein Tp0663,Tp0821,Tp0971 can induce the production of proinflammatory cytokines (CKs) IL-6 and IL-1 尾 by macrophages. By observing the effects of TLR2 antibody, CD14 antibody and NF- kappa B specific inhibitor pyrrolidine dithiocarbamate (PDTC) on the production of CKs in macrophages induced by three membrane lipoproteins, the effects of these membrane lipoproteins on the pre-production of CKs and TLR2, were studied. The signal transduction pathway mediated by CD14 and NF- 魏 B is related. Methods: 1 the gene sequences of selected Tp0663,Tp0821 and Tp0971 were obtained by Genbank, and the target fragment was amplified by PCR using genomic DNA of Tp Nichols strain as template. The recombinant plasmid pET28a () / Tp0663, was subcloned into prokaryotic expression vector pET28a () to construct the recombinant plasmid pET28a () / Tp0663,. PET28a () / Tp0821 and pET28a () / Tp0971, were digested by PCR, and identified by sequencing. They were transformed into the host strain E.coli Rosseta and induced by IPTG. The expression products were analyzed and identified by SDS-PAGE and Western blot, the recombinant protein was purified by Ni-NTA affinity chromatography, the protein concentration was determined by BCA, and the protein concentration was determined by Detoxi-Gel?. Endotoxin was removed from the recombinant protein and the endotoxin content was measured by Limulus lysate. 2 (PMA) induced the transformation of human monocytes THP-1 into macrophages, and the macrophages were stimulated by Tp0663,Tp0821 and Tp0971 recombinant proteins. The proinflammatory cytokines IL-6 and IL-1 尾 were detected by ELISA double antibody sandwich method. After macrophages were pretreated with TLR2 antibody, CD14 antibody and PDTC, the macrophages were stimulated with Tp0663,Tp0821 and p0971 recombinant protein, respectively. The effects of TLR2 antibody, CD14 antibody and PDTC on IL-6 and IL-1 尾 induced by recombinant protein were analyzed by ELISA. Results: the recombinant plasmid was confirmed to be the target gene by restriction endonuclease digestion and sequencing. The result of sequencing was identical with the login sequence of Genbank. The results of SDS-PAGE showed that under the induction of IPTG, a recombinant protein with relative molecular weight of 34kDa (Tp0663), 36 kDa (Tp0821) and 29 kDa (Tp0971) was expressed respectively. The target protein existed in the form of soluble and inclusion bodies in the cell. After purification with Ni-NTA affinity, the purity of the protein was over 95%. The results showed that it was consistent with the size of the target protein. The recombinant protein was removed by lipopolysaccharide (LPS), and the endotoxin was less than 0.04 EU / mL by Limulus lysate (Limulus lysate). THP-1 cells were transformed into macrophages by PMA stimulation. Different concentrations of recombinant protein (0.5 渭 g / ml~ 10 渭 g / ml) could significantly induce the production of IL-6 and IL-1 尾 by macrophages. When the concentration of recombinant protein was higher than 3 渭 g / ml (Tp0663) and 5 渭 g / ml (Tp0821 and Tp0971), the production of IL-6 and IL-1 尾 did not increase significantly. The production of IL-6 induced by Tp0663, Tp0821 and Tp0971 decreased to 66.0% after treatment with TLR 2 antibody. The production of IL-1 尾 decreased to 63.0%, 62.0% and 62.0%, respectively, at 64.0% and 60.0%, respectively. After treatment with CD14 antibody, the production of IL-6 decreased to 71.0%, 73.0% and 69.0%, and the production of IL-1 尾 decreased to 69.0%, 70.0% and 66.0%, respectively, and the production of IL-1 尾 decreased to 71.0%, 73.0% and 69.0%, respectively. The production of IL-6 and IL-1 尾 decreased to 54.0%, 55.0% and 56.0%, respectively, and IL-1 尾 production decreased to 51.0%, 50.0% and 52.0% after combined treatment of TLR2 and CD14 antibody, respectively, and the production of IL-1 尾 decreased to 51.0%, 50.0% and 52.0%, respectively. The production of IL-6 and IL-1 尾 decreased to less than 20% after treatment with NF- kappa B specific inhibitor PDTC, and there was significant difference between each treatment group and the control group (P0.05). Conclusions: 1. The recombinant proteins of Tp0663,Tp0821 and Tp0971 can induce the production of CKs IL-6 and IL-1 尾 in macrophages. The pre-inflammatory CKs induced by Tp0663,Tp0821 and Tp0971 recombinant proteins is related to the signal transduction pathways involved in TLR2, CD14 and NF- 魏 B.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R377
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