鼠伤寒沙门氏菌EF-Tu蛋白与宿主（THP-1）蛋白相互作用的筛

发布时间：2018-01-29 19:42

本文关键词： EF-Tu 鼠伤寒沙门菌 GST-Pull down 蛋白相互作用 THP-1　出处：《山东农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：沙门氏菌是一类对人和畜禽健康构成极大危害的革兰氏阴性致病菌,其中鼠伤寒沙门菌的感染率居沙门菌感染的首位,在世界范围内是一种常见的食品污染源。据调查,在引起机体肠炎的沙门氏菌中,鼠伤寒沙门氏菌占主要部分,且感染、发病率呈明显增高趋势。鼠伤寒沙门菌作为兼性胞内菌,能够在宿主单核吞噬细胞系统内生长、繁殖并随之迁移,避免抗体作用,而形成的SCV小泡也为细菌提供了一个安全稳定的寄生环境,这确保沙门氏菌能在机体持续存在。目前,细菌EF-Tu蛋白已被证实具有多种生物学功能,在多种菌属中均具有诱导宿主免疫应答的能力。提出假设:在鼠伤寒沙门菌感染宿主过程中,EF-Tu有可能作为一种重要的免疫原被宿主细胞所识别和捕获,在细菌与宿主细胞之间发挥作用。鼠伤寒沙门菌对小鼠的致病机理以及机体对鼠伤寒沙门菌的防御机制了解较多,而在人体内、体外研究较少,为进一步了解鼠伤寒沙门菌对人体的致病机制,本研究将鼠伤寒沙门菌EF-Tu蛋白作为“诱饵”,人单核吞噬细胞(THP-1)作为宿主,运用GST-pull down方法筛选出与EF-Tu相关联的细胞蛋白,并进行验证,以开展沙门氏菌—宿主细胞相互作用的研究,为阐明沙门氏菌致病分子机制提供依据。实验由以下部分构成:1.原核表达EF-Tu蛋白以鼠伤寒沙门菌基因组为模板扩增tufA基因片段,完成原核表达载体的构建。将重组质粒pGEX-6p-1-tufA和pET30a-tufA转入E.coli中,1mM IPTG诱导表达,SDSPAGE电泳和Western blot进行检验。结果显示原核表达蛋白GST-EF-Tu和His-EF-Tu与理论值相近。2.EF-Tu蛋白单克隆抗体制备原核蛋白His-EF-Tu免疫BALB/c小鼠后,当抗体效价达到单克隆抗体制备的标准,通过细胞融合、间接ELISA筛选、5次亚克隆获得5组稳定分泌单克隆抗体的阳鼠伤寒沙门氏菌翻译延伸因子ef-tu蛋白与宿主(thp-1)蛋白相互作用的筛选、验证和功能研究性杂交瘤细胞,再扩大培养收集细胞培养上清作为单克隆抗体,这5组单抗的亚型均为igg1/к。将免疫原蛋白his-ef-tu、鼠伤寒沙门氏菌菌体蛋白/大肠杆菌菌体蛋白、重组蛋白gst-ef-tu及金黄色葡萄球菌菌体蛋白(阴性对照)为样品进行western-blot分析,单克隆抗体作为一抗,在dab显色后分别得到了大小与理论值相符的条带,ef-tu单抗具有特异性。3.鼠伤寒沙门氏菌ef-tu的表面定位活化培养鼠伤寒沙门氏菌至对数期后,取少量菌液作待检样品。以ef-tumab/pcab为工具,依次完成间接免疫荧光实验、流式细胞仪分析和补体中和试验对ef-tu进行定位,结果发现ef-tu存在于鼠伤寒沙门氏菌菌体表面。4.筛选与ef-tu相互作用的宿主细胞蛋白制备新鲜的宿主细胞蛋白与纯化后的ef-tu蛋白共孵育,设立对照组。将孵育后的产物用pbs(ph7.4)轻柔洗涤,除去杂蛋白后,从gstresin上洗脱,获得蛋白复合物。为观察是否存在与ef-tu蛋白相互作用的宿主细胞细胞蛋白,将蛋白复合物进行sds-page电泳,考马斯亮蓝染色,甲醇脱色。结果发现凝胶中多出两条蛋白条带。将凝胶中的蛋白质样品送去公司进行质谱测序,反馈信息得到两个蛋白,即pkm2和eef1α1。5.验证ef-tu与两种细胞蛋白存在相互作用提取宿主细胞rna,反转录为cdna作为模板,扩增pkm2和eef1α1基因片段,构建宿主细胞原核表达载体pet28a-pkm2、pet30a-eef1α1。将重组质粒分别转入e.coli中进行原核表达,验证表达成功后,大量表达并用ni柱进行his-pkm2和his-eef1α1蛋白的纯化。将新鲜的ef-tu蛋白分别与纯化后的his-pkm2和his-eef1α1共孵育,设立对照组。将孵育后的产物洗涤,除去杂蛋白后,从ni柱上洗脱,获得蛋白复合物进行westernblot分析。结果发现:dab显色后,pvdf膜上显出特异性条带,而对照组呈阴性反应。随后构建真核表达载体pdsred1-n1-tufa,并转染hela细胞,以ifa检测显示:ef-tu蛋白在细胞内成功表达。设立两个转染组,完成后对山东农业大学硕士学位论文Hela细胞内的pkM2、eEF1α1蛋白分别进行荧光染色,于激光共聚焦显微镜观察。结果表明,EF-Tu蛋白与pkM2、eEF1α1蛋白分别共定位在细胞质,这在空间上为两者发生联系提供佐证。6.鼠伤寒沙门菌侵染宿主细胞对pkM2和eEF1α1表达量的影响以1:100的比例将鼠伤寒沙门菌接种到细胞板,与宿主细胞共培养,使鼠伤寒沙门菌侵染宿主。在不同时间段取出细胞样品,以GADPH为参照,进行荧光定量检测。结果发现随着鼠伤寒沙门菌侵染细胞的时间增加,pkM2和eEF1α1的表达量减少。
[Abstract]:Salmonella is a kind of human and animal health and endangers the gram negative pathogens, including Salmonella typhimurium infection rate in Salmonella infection in the first place, in the world of food is one of common sources. According to the survey, in the body caused by Salmonella enteritidis and Salmonella typhimurium accounted for the main part, and the infection incidence rate was significantly higher trend. Salmonella typhimurium as a facultative intracellular bacterium that can host mononuclear phagocyte system in growth, reproduction and migrated, avoid the antibody effect and the formation of vesicles, SCV also provides a safe and stable environment to ensure the parasitic bacteria, Salmonella bacteria can continuously exist. At present, the bacterial EF-Tu protein have been shown to have a variety of biological functions, has the ability to induce host immune response in a variety of bacteria of the genus. Put forward the hypothesis: in the rat typhoid Salmonella infection Host process, EF-Tu may be used as an important immunogen by the host cell recognition and capture, play a role in between bacteria and host cells. The defense mechanism of pathogenic mechanism of Salmonella typhimurium in mice and immunity to Salmonella typhimurium more understanding, and in the human body, less research in vitro, in order to further understand the pathogenesis the mechanism of Salmonella typhimurium on the human body, the Salmonella typhimurium EF-Tu protein as bait, human mononuclear phagocytes (THP-1) as the host, the use of GST-pull down methodscreed associated with EF-Tu cell protein, and verify, to carry out research on Salmonella - host cell interactions, provide the basis for clarifying the mechanism's pathogenic Salmonella. The experiment consists of the following parts: 1. the prokaryotic expression of EF-Tu protein in Salmonella typhimurium genome as template to amplify tufA gene fragments, end To construct prokaryotic expression vector. The recombinant plasmid pGEX-6p-1-tufA and pET30a-tufA into E.coli and 1mM IPTG expression induced by SDSPAGE electrophoresis and Western blot, were tested. The results showed that the prokaryotic expression of GST-EF-Tu and His-EF-Tu with the theoretical value of similar preparation of monoclonal antibody against.2.EF-Tu protein prokaryotic protein His-EF-Tu immune BALB/c mice, when the monoclonal antibody titer reached antibody preparation standard, through cell fusion, indirect ELISA screening, Salmonella typhimurium translation sapientis subcloned 5 times to obtain 5 groups of stably secreting monoclonal antibody elongation factor EF-Tu protein and host (THP-1) screening of protein interactions, and functional verification of hybridoma cells, and then expand the culture supernatants were collected as monoclonal these 5 groups of antibodies, monoclonal antibody subtypes were igg1/. The kappa immunogenic protein his-ef-tu, Salmonella typhimurium / Escherichia coli protein Bacterial protein, recombinant protein gst-ef-tu and Staphylococcus aureus protein (negative control) were analyzed by Western-blot, monoclonal antibody as the first antibody in DAB color size were obtained and the theoretical value of strip line, surface localization of EF-Tu monoclonal antibody with specificity for.3. Salmonella typhimurium EF-Tu activation of cultured rat typhoid Salmonella in the logarithmic phase, take a small amount of bacteria liquid sample. Using ef-tumab/pcab as the tool, in order to complete the indirect immunofluorescence assay, flow cytometric analysis and complement neutralization test results showed that the localization of EF-Tu, EF-Tu in Salmonella typhimurium.4. screening cell surface interaction between EF-Tu and host cell proteins preparation of fresh host cell protein and purified EF-Tu protein were incubated, the establishment of the control group. After incubation of the product with PBS (pH7.4) gentle washing, removed proteins, from Gstresin was obtained protein complex. To observe the existence of host cell protein interacting with EF-Tu protein, the protein complexes were analyzed by SDS-PAGE electrophoresis, Coomassie brilliant blue staining Kaumas, methanol. The results showed that two protein bands in the gel. The gel is a protein in the samples sent to the company by mass spectrometry sequencing. The feedback information of two proteins, namely pkm2 and EEF1 alpha 1.5. verification EF-Tu and two kinds of cell protein interaction between host cells extracted RNA, reverse transcription cDNA as template, pkm2 amplification and EEF1 alpha 1 gene fragments of host cells, construct the prokaryotic expression vector pet28a-pkm2, pet30a-eef1 alpha 1. recombinant plasmid was transformed into expression of prokaryotic E.coli, verify the expression after the success of a large number of expression and purification of 1 protein using Ni column his-pkm2 and his-eef1 alpha. Fresh EF-Tu proteins were purified with his-pkm2 and his-ee Alpha 1 F1 co incubation, the establishment of the control group. After incubation of the product of washing, removing impurity protein after elution from the Ni column, to obtain protein complexes were analyzed by Westernblot. The results showed that: DAB color, PVDF film shows a specific band, while the control group was negative reaction. Then construct the eukaryotic expression vector pdsred1-n1-tufa, and transfected into HeLa cells, with IFA detection: the successful expression of EF-Tu protein in cells. The establishment of two transfection group, pkM2 of Shandong Agricultural University master's degree thesis in Hela cells after eEF1 alpha 1 protein were stained by laser confocal microscopy. The results showed that EF-Tu protein with pkM2, eEF1 alpha 1 proteins were co localized in the cytoplasm, which in the space for the two links provide evidence.6. of Salmonella typhimurium infected host cells of pkM2 and eEF1 alpha 1 expression as the ratio of 1:100 with Salmonella typhimurium A cell plate, co cultured with host cells to Salmonella typhimurium infected host. Take out the cell samples at different time, with GADPH as a reference, fluorescence quantitative detection. Results showed that Salmonella typhimurium infected cells with the time increasing, the expression of pkM2 and eEF1 alpha 1 reduced.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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