本文選題：變形鏈球菌 + 細胞外蛋白　； 參考：《中國人民解放軍醫(yī)學院》2016年碩士論文

【摘要】：變形鏈球菌是口腔內的重要致齲菌之一,也被證明與許多全身系統(tǒng)性疾病有密切的關系。細胞外蛋白往往含有致病或毒性因子,與疾病發(fā)病機制密切相關,對病理研究和抗生素開發(fā)等具有重要意義。蛋白質組即是在特定時間細胞內的所有蛋白質,蛋白質組學是從整體水平研究動態(tài)變化的蛋白質組成、修飾狀態(tài)和表達水平。從而了解蛋白質之間的相互作用。細菌的細胞外蛋白質組學已經(jīng)是一個相當成熟的研究領域,并取得了重要的研究成果。我們分三部分內容對差異致齲性變形鏈球菌臨床分離株細胞外蛋白質組學進行研究,希望從蛋白質水平探討變形鏈球菌的致齲機理,為臨床齲病防治提供幫助。第一部分致齲模型建立-羥基磷灰石粘附實驗,大鼠致齲模型實驗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,致使其具有強的致齲性。而粘附能力,是變鏈菌致齲的基礎。我們首先通過唾液包被的羥基磷灰石粘附實驗,對課題組前期分離鑒定的5株變形鏈球菌臨床分離株進行鑒定,發(fā)現(xiàn)各臨床菌株之間的粘附能力差異顯著,其中5號菌株粘附能力最強,4號菌株最弱。研究結果驗證了變形鏈球菌臨床分離株致齲特性的差異性,同時也建立了檢測變形鏈球菌致齲特性的體外實驗方法，為后續(xù)蛋白質致齲機制研究提供了手段;實驗二Wistar大鼠致齲模型建立我們建立了大鼠致齲模型,使用5號高致齲菌株作為實驗組用菌涂布于大鼠口腔內,對照組涂布生理鹽水。經(jīng)過兩個月的實驗周期,結果顯示：實驗組大鼠磨牙成功形成可觀察的齲壞,而對照組則只呈現(xiàn)陰性結果。動物實驗是驗證科學結論的金標準,我們在本實驗平臺,建立了標準的,可重復的大鼠致齲模型,為后續(xù)實驗夯實了基礎：第二部分 差異致齲性變鏈菌細胞外比較蛋白質組學研究我們在本實驗室條件下,測定了變形鏈球菌生長曲線。實驗提取了變形鏈球菌對數(shù)生長末期的培養(yǎng)上清,通過TCA沉淀法得到變鏈菌的全細胞外蛋白。對其進行了雙向電泳,及后期的質譜分析。通過雙向電泳我們比較了18個存在表達差異的點白點。質譜分析后,我們最后鎖定了S1 (3OS ribosomal protein S1), SacB (levansucrase precursor)兩個差異蛋白。他們在高低致齲性變形鏈菌細胞外蛋白雙向電泳中顯著差異表達,可能是導致上述臨床菌株致齲差異的關鍵因素。第三部分 變形鏈球菌菌S1, SacB蛋白的表達純化我們首先在基因水平對S1, sacB基因進行了檢測。qRT-PCR結果顯示S1,SacB在高致齲菌轉錄過程中顯著高表達,證實了我們雙向電泳結果分析的可信性。接著,通過原核表達系統(tǒng),我們構建了pET-28a-S1, pET-28a-SacB重組表達載體,并表達純化了S1蛋白。為后續(xù)的功能研究奠定了基礎。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R781.1
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本文編號：2102656