【摘要】： 目的:探討梅毒螺旋體(Treponema pallidum, Tp)膜脂蛋白Tp0663、Tp0821、Tp0971能否誘導(dǎo)巨噬細(xì)胞產(chǎn)生前炎癥細(xì)胞因子(CKs)IL-6和IL-1β;通過觀察TLR2抗體、CD14抗體、NF-κB特異抑制劑二硫代氨基甲酸吡咯烷(PDTC)對三種膜脂蛋白誘導(dǎo)巨噬細(xì)胞產(chǎn)生CKs的影響,研究這些膜脂蛋白誘導(dǎo)產(chǎn)生前炎癥CKs是否與TLR2、CD14及NF-κB介導(dǎo)的信號轉(zhuǎn)導(dǎo)途徑有關(guān)。 方法:①通過Genbank獲取選Tp0663、Tp0821和Tp0971的基因序列,以Tp Nichols株基因組DNA為模板,PCR擴(kuò)增目的片段,將其亞克隆至原核表達(dá)載體pET28a(+)中構(gòu)建重組質(zhì)粒pET28a(+)/Tp0663、pET28a(+)/Tp0821和pET28a(+)/Tp0971,經(jīng)PCR、雙酶切、測序鑒定后將其轉(zhuǎn)化至表達(dá)宿主菌E.coli Rosseta中,IPTG誘導(dǎo)表達(dá)。采用SDS-PAGE和Western blot分析和鑒定表達(dá)產(chǎn)物;Ni-NTA親和層析柱純化重組蛋白,BCA法測定蛋白濃度;Detoxi-Gel?內(nèi)毒素去除膠去除重組蛋白中的內(nèi)毒素,經(jīng)鱟試劑測定內(nèi)毒素含量。②佛波酯(PMA)誘導(dǎo)人單核細(xì)胞THP-1轉(zhuǎn)化為巨噬細(xì)胞,用Tp0663、Tp0821和Tp0971重組蛋白刺激巨噬細(xì)胞,ELISA雙抗體夾心法檢測誘生其前炎癥細(xì)胞因子IL-6和IL-1β的情況;用TLR2抗體、CD14抗體和PDTC預(yù)處理巨噬細(xì)胞后,用Tp0663、Tp0821和p0971重組蛋白分別刺激巨噬細(xì)胞,ELISA分析TLR2抗體、CD14抗體和PDTC對重組蛋白誘導(dǎo)巨噬細(xì)胞產(chǎn)生IL-6和IL-1β的影響。 結(jié)果:構(gòu)建的重組質(zhì)粒經(jīng)酶切和測序鑒定證實插入片段為目的基因,測序結(jié)果與Genbank上登錄序列完全一致;SDS-PAGE結(jié)果顯示,在IPTG誘導(dǎo)下,重組表達(dá)菌分別表達(dá)了一相對分子量(Mr)約為34kDa(Tp0663)和36 kDa(Tp0821),29 kDa(Tp0971)的重組蛋白,目的蛋白在菌體細(xì)胞內(nèi)以可溶性和包涵體形式存在,經(jīng)Ni-NTA親和純化后,純度在95%以上,Western blot結(jié)果顯示其與目的蛋白大小相符;內(nèi)毒素去除膠處理重組蛋白,經(jīng)鱟試劑檢測內(nèi)毒素小于0.04EU/mL;THP-1細(xì)胞經(jīng)PMA刺激轉(zhuǎn)化為巨噬細(xì)胞,不同濃度的重組蛋白(0.5μg/ml～10μg/ml)能明顯誘導(dǎo)巨噬細(xì)胞產(chǎn)生IL-6和IL-1β;當(dāng)重組蛋白濃度高于3μg/ml(Tp0663)和5μg/ml(Tp0821和Tp0971)時,IL-6和IL-1β產(chǎn)生的量無明顯增加。TLR2抗體處理后,Tp0663, Tp0821和Tp0971誘導(dǎo)IL-6的產(chǎn)生量分別降至66.0%、64.0%和60.0%,IL-1β的產(chǎn)生量分別降至63.0%、62.0%和62.0%;經(jīng)CD14抗體處理后, IL-6的產(chǎn)生量分別降至71.0%、73.0%和69.0%,IL-1β的產(chǎn)生量分別降至69.0%、70.0%和66.0%,經(jīng)TLR2和CD14抗體聯(lián)合處理后IL-6的產(chǎn)生量分別降至54.0%、55.0%和56.0%,IL-1β的產(chǎn)生量分別降至51.0%、50.0%和52.0%,NF-κB特異性抑制劑PDTC處理后IL-6和IL-1β的產(chǎn)生量分別降至20%以下,各處理組與對照組(同型抗體)比較(P0.05)有明顯差異。 結(jié)論: 1. Tp0663、Tp0821和Tp0971重組蛋白能誘導(dǎo)巨噬細(xì)胞產(chǎn)生前炎癥CKs IL-6和IL-1β; 2. Tp0663、Tp0821和Tp0971重組蛋白誘導(dǎo)巨噬細(xì)胞產(chǎn)生前炎癥CKs與TLR2和CD14及NF-κB參與的信號轉(zhuǎn)導(dǎo)通路有關(guān)。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R377

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