差异致龋性变形链球菌临床分离株细胞外比较蛋白质组学研究

发布时间：2018-07-06 11:37

  本文选题：变形链球菌 + 细胞外蛋白　； 参考：《中国人民解放军医学院》2016年硕士论文

【摘要】：变形链球菌是口腔内的重要致龋菌之一,也被证明与许多全身系统性疾病有密切的关系。细胞外蛋白往往含有致病或毒性因子,与疾病发病机制密切相关,对病理研究和抗生素开发等具有重要意义。蛋白质组即是在特定时间细胞内的所有蛋白质,蛋白质组学是从整体水平研究动态变化的蛋白质组成、修饰状态和表达水平。从而了解蛋白质之间的相互作用。细菌的细胞外蛋白质组学已经是一个相当成熟的研究领域,并取得了重要的研究成果。我们分三部分内容对差异致龋性变形链球菌临床分离株细胞外蛋白质组学进行研究,希望从蛋白质水平探讨变形链球菌的致龋机理,为临床龋病防治提供帮助。第一部分致龋模型建立-羟基磷灰石粘附实验,大鼠致龋模型实验一唾液包被的羟基磷灰石致龋模型建立变链菌粘附牙面,并最终形成牙菌斑生物膜,之后其耐酸产酸能力,致使其具有强的致龋性。而粘附能力,是变链菌致龋的基础。我们首先通过唾液包被的羟基磷灰石粘附实验,对课题组前期分离鉴定的5株变形链球菌临床分离株进行鉴定,发现各临床菌株之间的粘附能力差异显著,其中5号菌株粘附能力最强,4号菌株最弱。研究结果验证了变形链球菌临床分离株致龋特性的差异性,同时也建立了检测变形链球菌致龋特性的体外实验方法，为后续蛋白质致龋机制研究提供了手段;实验二Wistar大鼠致龋模型建立我们建立了大鼠致龋模型,使用5号高致龋菌株作为实验组用菌涂布于大鼠口腔内,对照组涂布生理盐水。经过两个月的实验周期,结果显示：实验组大鼠磨牙成功形成可观察的龋坏,而对照组则只呈现阴性结果。动物实验是验证科学结论的金标准,我们在本实验平台,建立了标准的,可重复的大鼠致龋模型,为后续实验夯实了基础：第二部分 差异致龋性变链菌细胞外比较蛋白质组学研究我们在本实验室条件下,测定了变形链球菌生长曲线。实验提取了变形链球菌对数生长末期的培养上清,通过TCA沉淀法得到变链菌的全细胞外蛋白。对其进行了双向电泳,及后期的质谱分析。通过双向电泳我们比较了18个存在表达差异的点白点。质谱分析后,我们最后锁定了S1 (3OS ribosomal protein S1), SacB (levansucrase precursor)两个差异蛋白。他们在高低致龋性变形链菌细胞外蛋白双向电泳中显著差异表达,可能是导致上述临床菌株致龋差异的关键因素。第三部分 变形链球菌菌S1, SacB蛋白的表达纯化我们首先在基因水平对S1, sacB基因进行了检测。qRT-PCR结果显示S1,SacB在高致龋菌转录过程中显著高表达,证实了我们双向电泳结果分析的可信性。接着,通过原核表达系统,我们构建了pET-28a-S1, pET-28a-SacB重组表达载体,并表达纯化了S1蛋白。为后续的功能研究奠定了基础。
[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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R781.1
，

本文编号：2102656