本文關(guān)鍵詞： EF-Tu 鼠傷寒沙門菌 GST-Pull down 蛋白相互作用 THP-1　出處：《山東農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：沙門氏菌是一類對人和畜禽健康構(gòu)成極大危害的革蘭氏陰性致病菌,其中鼠傷寒沙門菌的感染率居沙門菌感染的首位,在世界范圍內(nèi)是一種常見的食品污染源。據(jù)調(diào)查,在引起機體腸炎的沙門氏菌中,鼠傷寒沙門氏菌占主要部分,且感染、發(fā)病率呈明顯增高趨勢。鼠傷寒沙門菌作為兼性胞內(nèi)菌,能夠在宿主單核吞噬細胞系統(tǒng)內(nèi)生長、繁殖并隨之遷移,避免抗體作用,而形成的SCV小泡也為細菌提供了一個安全穩(wěn)定的寄生環(huán)境,這確保沙門氏菌能在機體持續(xù)存在。目前,細菌EF-Tu蛋白已被證實具有多種生物學(xué)功能,在多種菌屬中均具有誘導(dǎo)宿主免疫應(yīng)答的能力。提出假設(shè):在鼠傷寒沙門菌感染宿主過程中,EF-Tu有可能作為一種重要的免疫原被宿主細胞所識別和捕獲,在細菌與宿主細胞之間發(fā)揮作用。鼠傷寒沙門菌對小鼠的致病機理以及機體對鼠傷寒沙門菌的防御機制了解較多,而在人體內(nèi)、體外研究較少,為進一步了解鼠傷寒沙門菌對人體的致病機制,本研究將鼠傷寒沙門菌EF-Tu蛋白作為“誘餌”,人單核吞噬細胞(THP-1)作為宿主,運用GST-pull down方法篩選出與EF-Tu相關(guān)聯(lián)的細胞蛋白,并進行驗證,以開展沙門氏菌—宿主細胞相互作用的研究,為闡明沙門氏菌致病分子機制提供依據(jù)。實驗由以下部分構(gòu)成:1.原核表達EF-Tu蛋白以鼠傷寒沙門菌基因組為模板擴增tufA基因片段,完成原核表達載體的構(gòu)建。將重組質(zhì)粒pGEX-6p-1-tufA和pET30a-tufA轉(zhuǎn)入E.coli中,1mM IPTG誘導(dǎo)表達,SDSPAGE電泳和Western blot進行檢驗。結(jié)果顯示原核表達蛋白GST-EF-Tu和His-EF-Tu與理論值相近。2.EF-Tu蛋白單克隆抗體制備原核蛋白His-EF-Tu免疫BALB/c小鼠后,當(dāng)抗體效價達到單克隆抗體制備的標(biāo)準(zhǔn),通過細胞融合、間接ELISA篩選、5次亞克隆獲得5組穩(wěn)定分泌單克隆抗體的陽鼠傷寒沙門氏菌翻譯延伸因子ef-tu蛋白與宿主(thp-1)蛋白相互作用的篩選、驗證和功能研究性雜交瘤細胞,再擴大培養(yǎng)收集細胞培養(yǎng)上清作為單克隆抗體,這5組單抗的亞型均為igg1/к。將免疫原蛋白his-ef-tu、鼠傷寒沙門氏菌菌體蛋白/大腸桿菌菌體蛋白、重組蛋白gst-ef-tu及金黃色葡萄球菌菌體蛋白(陰性對照)為樣品進行western-blot分析,單克隆抗體作為一抗,在dab顯色后分別得到了大小與理論值相符的條帶,ef-tu單抗具有特異性。3.鼠傷寒沙門氏菌ef-tu的表面定位活化培養(yǎng)鼠傷寒沙門氏菌至對數(shù)期后,取少量菌液作待檢樣品。以ef-tumab/pcab為工具,依次完成間接免疫熒光實驗、流式細胞儀分析和補體中和試驗對ef-tu進行定位,結(jié)果發(fā)現(xiàn)ef-tu存在于鼠傷寒沙門氏菌菌體表面。4.篩選與ef-tu相互作用的宿主細胞蛋白制備新鮮的宿主細胞蛋白與純化后的ef-tu蛋白共孵育,設(shè)立對照組。將孵育后的產(chǎn)物用pbs(ph7.4)輕柔洗滌,除去雜蛋白后,從gstresin上洗脫,獲得蛋白復(fù)合物。為觀察是否存在與ef-tu蛋白相互作用的宿主細胞細胞蛋白,將蛋白復(fù)合物進行sds-page電泳,考馬斯亮藍染色,甲醇脫色。結(jié)果發(fā)現(xiàn)凝膠中多出兩條蛋白條帶。將凝膠中的蛋白質(zhì)樣品送去公司進行質(zhì)譜測序,反饋信息得到兩個蛋白,即pkm2和eef1α1。5.驗證ef-tu與兩種細胞蛋白存在相互作用提取宿主細胞rna,反轉(zhuǎn)錄為cdna作為模板,擴增pkm2和eef1α1基因片段,構(gòu)建宿主細胞原核表達載體pet28a-pkm2、pet30a-eef1α1。將重組質(zhì)粒分別轉(zhuǎn)入e.coli中進行原核表達,驗證表達成功后,大量表達并用ni柱進行his-pkm2和his-eef1α1蛋白的純化。將新鮮的ef-tu蛋白分別與純化后的his-pkm2和his-eef1α1共孵育,設(shè)立對照組。將孵育后的產(chǎn)物洗滌,除去雜蛋白后,從ni柱上洗脫,獲得蛋白復(fù)合物進行westernblot分析。結(jié)果發(fā)現(xiàn):dab顯色后,pvdf膜上顯出特異性條帶,而對照組呈陰性反應(yīng)。隨后構(gòu)建真核表達載體pdsred1-n1-tufa,并轉(zhuǎn)染hela細胞,以ifa檢測顯示:ef-tu蛋白在細胞內(nèi)成功表達。設(shè)立兩個轉(zhuǎn)染組,完成后對山東農(nóng)業(yè)大學(xué)碩士學(xué)位論文Hela細胞內(nèi)的pkM2、eEF1α1蛋白分別進行熒光染色,于激光共聚焦顯微鏡觀察。結(jié)果表明,EF-Tu蛋白與pkM2、eEF1α1蛋白分別共定位在細胞質(zhì),這在空間上為兩者發(fā)生聯(lián)系提供佐證。6.鼠傷寒沙門菌侵染宿主細胞對pkM2和eEF1α1表達量的影響以1:100的比例將鼠傷寒沙門菌接種到細胞板,與宿主細胞共培養(yǎng),使鼠傷寒沙門菌侵染宿主。在不同時間段取出細胞樣品,以GADPH為參照,進行熒光定量檢測。結(jié)果發(fā)現(xiàn)隨著鼠傷寒沙門菌侵染細胞的時間增加,pkM2和eEF1α1的表達量減少。
[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.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

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