本文選題：變形鏈球菌 + 細(xì)胞外蛋白��； 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2016年碩士論文

【摘要】：變形鏈球菌是口腔內(nèi)的重要致齲菌之一,也被證明與許多全身系統(tǒng)性疾病有密切的關(guān)系。細(xì)胞外蛋白往往含有致病或毒性因子,與疾病發(fā)病機(jī)制密切相關(guān),對(duì)病理研究和抗生素開發(fā)等具有重要意義。蛋白質(zhì)組即是在特定時(shí)間細(xì)胞內(nèi)的所有蛋白質(zhì),蛋白質(zhì)組學(xué)是從整體水平研究動(dòng)態(tài)變化的蛋白質(zhì)組成、修飾狀態(tài)和表達(dá)水平。從而了解蛋白質(zhì)之間的相互作用。細(xì)菌的細(xì)胞外蛋白質(zhì)組學(xué)已經(jīng)是一個(gè)相當(dāng)成熟的研究領(lǐng)域,并取得了重要的研究成果。我們分三部分內(nèi)容對(duì)差異致齲性變形鏈球菌臨床分離株細(xì)胞外蛋白質(zhì)組學(xué)進(jìn)行研究,希望從蛋白質(zhì)水平探討變形鏈球菌的致齲機(jī)理,為臨床齲病防治提供幫助。第一部分致齲模型建立-羥基磷灰石粘附實(shí)驗(yàn),大鼠致齲模型實(shí)驗(yàn)一唾液包被的羥基磷灰石致齲模型建立變鏈菌粘附牙面,并最終形成牙菌斑生物膜,之后其耐酸產(chǎn)酸能力,致使其具有強(qiáng)的致齲性。而粘附能力,是變鏈菌致齲的基礎(chǔ)。我們首先通過(guò)唾液包被的羥基磷灰石粘附實(shí)驗(yàn),對(duì)課題組前期分離鑒定的5株變形鏈球菌臨床分離株進(jìn)行鑒定,發(fā)現(xiàn)各臨床菌株之間的粘附能力差異顯著,其中5號(hào)菌株粘附能力最強(qiáng),4號(hào)菌株最弱。研究結(jié)果驗(yàn)證了變形鏈球菌臨床分離株致齲特性的差異性,同時(shí)也建立了檢測(cè)變形鏈球菌致齲特性的體外實(shí)驗(yàn)方法，為后續(xù)蛋白質(zhì)致齲機(jī)制研究提供了手段;實(shí)驗(yàn)二Wistar大鼠致齲模型建立我們建立了大鼠致齲模型,使用5號(hào)高致齲菌株作為實(shí)驗(yàn)組用菌涂布于大鼠口腔內(nèi),對(duì)照組涂布生理鹽水。經(jīng)過(guò)兩個(gè)月的實(shí)驗(yàn)周期,結(jié)果顯示：實(shí)驗(yàn)組大鼠磨牙成功形成可觀察的齲壞,而對(duì)照組則只呈現(xiàn)陰性結(jié)果。動(dòng)物實(shí)驗(yàn)是驗(yàn)證科學(xué)結(jié)論的金標(biāo)準(zhǔn),我們?cè)诒緦?shí)驗(yàn)平臺(tái),建立了標(biāo)準(zhǔn)的,可重復(fù)的大鼠致齲模型,為后續(xù)實(shí)驗(yàn)夯實(shí)了基礎(chǔ)：第二部分 差異致齲性變鏈菌細(xì)胞外比較蛋白質(zhì)組學(xué)研究我們?cè)诒緦?shí)驗(yàn)室條件下,測(cè)定了變形鏈球菌生長(zhǎng)曲線。實(shí)驗(yàn)提取了變形鏈球菌對(duì)數(shù)生長(zhǎng)末期的培養(yǎng)上清,通過(guò)TCA沉淀法得到變鏈菌的全細(xì)胞外蛋白。對(duì)其進(jìn)行了雙向電泳,及后期的質(zhì)譜分析。通過(guò)雙向電泳我們比較了18個(gè)存在表達(dá)差異的點(diǎn)白點(diǎn)。質(zhì)譜分析后,我們最后鎖定了S1 (3OS ribosomal protein S1), SacB (levansucrase precursor)兩個(gè)差異蛋白。他們?cè)诟叩椭慢x性變形鏈菌細(xì)胞外蛋白雙向電泳中顯著差異表達(dá),可能是導(dǎo)致上述臨床菌株致齲差異的關(guān)鍵因素。第三部分 變形鏈球菌菌S1, SacB蛋白的表達(dá)純化我們首先在基因水平對(duì)S1, sacB基因進(jìn)行了檢測(cè)。qRT-PCR結(jié)果顯示S1,SacB在高致齲菌轉(zhuǎn)錄過(guò)程中顯著高表達(dá),證實(shí)了我們雙向電泳結(jié)果分析的可信性。接著,通過(guò)原核表達(dá)系統(tǒng),我們構(gòu)建了pET-28a-S1, pET-28a-SacB重組表達(dá)載體,并表達(dá)純化了S1蛋白。為后續(xù)的功能研究奠定了基礎(chǔ)。
[Abstract]:Streptococcus mutans is one of the most important cariogenic bacteria in oral cavity and has been proved to be closely related to many systemic diseases. Extracellular proteins often contain pathogenic or toxic factors, which are closely related to the pathogenesis of the disease, and have important significance for the study of pathology and the development of antibiotics. Proteome is all the proteins in the cell at a certain time. Proteomics is to study the dynamic changes of protein composition, modified state and expression level from the overall level. In order to understand the interaction between proteins. Extracellular proteomics of bacteria is a very mature research field, and has made important research results. We divided three parts to study the extracellular proteomics of Streptococcus mutans (Streptococcus mutans). We hope to explore the mechanism of Streptococcus mutans caries from protein level and to provide help for the prevention and treatment of clinical caries. The first part was the establishment of dental caries model-hydroxyapatite adhesion test, and the salivary coated hydroxyapatite caries model to establish dental surface adhesion of Streptococcus mutans, and finally to form dental plaque biofilm, and then its acid-resistant acid-producing ability. So that it has strong caries. The adhesion ability is the basis of Streptococcus mutans caries. Firstly, we identified 5 clinical strains of Streptococcus mutans by saliva coated hydroxyapatite adhesion test. The adhesion ability of strain 5 was the strongest, and strain 4 was the weakest. The results verified the difference of caries characteristics of Streptococcus mutans clinical isolates, and established an in vitro experimental method to detect the caries characteristics of Streptococcus mutans, which provided a means for further study on the mechanism of protein caries. Experiment 2: we established the caries model of Wistar rats. The experimental group was coated with bacteria in the oral cavity of rats and the control group was coated with normal saline. After two months of experiment, the results showed that the rat molars in the experimental group successfully formed observable caries, while the control group only showed negative results. Animal experiments are the gold standard for verifying scientific conclusions, and we have established a standard, repeatable rat caries model on this experimental platform. In order to lay a solid foundation for further experiments: the second part of the differential Streptococcus mutans extracellular comparative proteomics study we measured the growth curve of Streptococcus mutans under the conditions of our laboratory. The culture supernatant of Streptococcus mutans at the end of logarithmic growth was extracted and the whole extracellular protein of Streptococcus mutans was obtained by TCA precipitation. It was analyzed by two-dimensional electrophoresis and later mass spectrometry. By two-dimensional electrophoresis, we compared 18 white spots with different expression. After mass spectrometry analysis, we finally identified S1 (3 OS ribosomal protein S1) and SACB (levansucrase precursor) two differential proteins. Their distinct differential expression in two dimensional electrophoresis of extracellular proteins of Streptococcus mutans may be the key factor leading to the difference in caries induced by these clinical strains. In the third part, the expression and purification of S1, SacB protein of Streptococcus mutans were detected at the gene level. The results of qRT-PCR showed that S1 SacB was significantly overexpressed during the transcription of high caries causing bacteria. The reliability of our two-dimensional electrophoresis analysis was confirmed. Then, we constructed the recombinant expression vector pET-28a-S1, pET-28a-SacB by prokaryotic expression system, and expressed and purified S1 protein. It lays a foundation for further functional research.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R781.1
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本文編號(hào)：2102656