高、低致龋性变异链球菌临床分离株关键差异蛋白与差异sRNA的鉴定

发布时间：2018-09-01 16:40

【摘要】：龋病,作为一种慢性、多因素疾病影响着人类健康。变异链球菌作为口腔内首要致龋菌,致病机制复杂,与其耐酸性紧密相关。不同菌株间的变异链球菌致龋毒力可存在很大差异,这一发现逐渐引起关注。目前,相关的研究主要建立在差异蛋白质组学和通过高通量测序获取关键调控基因基础上。SELEX技术主要利用单链核苷酸文库筛选与靶物质特异性结合的核酸适配体(aptamer)。Aptamer又称“人工抗体”,识别模式与蛋白抗体相似,具有无免疫原性、识别精准、修饰方便等优势,作为新型分子探针应用。细胞消减SELEX可筛选出特异识别两种同源靶物质上的差异aptamer,如本课题组前期获得的识别高龋菌的aptamer-H19,在龋病易感性预测方面存在潜在应用价值。细菌非编码小RNA (small non-coding RNA,sRNA)则主要通过碱基配对结合靶mRNA,从而在转录后水平发挥调控致病基因的作用。采用高通量筛选技术结合生物信息学预测的方法成为近年来研究、获取sRNA信息的主流。sRNA在环境耐受、细菌毒力、耐药性等方面的作用也是本课题组的研究热点。课题组通过前期对高、低致龋性变异链球菌临床分离株的筛选和研究,获得了性质稳定的分别具有高、低致龋特性的变异链球菌临床分离株(菌株5和菌株4)。本实验目的是利用特异aptamer-H19钓取高、低致龋菌株间的差异蛋白质同时研究采用高通量技术得到的差异sRNA在耐酸反应中的变化情况。第一部分:高、低致龋性变异链球菌临床分离株差异蛋白的筛选和鉴定利用特异识别高致龋菌株的aptamer-H19结合Pull-down技术从细菌总蛋白中钓取差异蛋白,经SDS-PAGE电泳、银染、质谱鉴定,获得候选蛋白信息。制备GroEL多克隆抗体并合成多肽,采用Western Blot以及qRT-PCR观察在不同pH培养条件下菌株4、5中的表达水平。发现其中菌株4、5皆存在表达差异且酸性条件下差异更加显著。第二部分:高、低致龋性变异链球菌临床分离株差异sRNA的筛选和鉴定提取菌株4、5总RNA,分离sRNA、构建文库,高通量筛选出已知的差异sRNA SpR19; qRT-PCR观察高、低致龋性变异链球菌临床分离株在不同pH情况下SpR19的表达变化。无论正常还是酸性环境下高致龋菌表达量都下调,与GroEL mRNA表现相反。经过生物信息学分析,发现SpR19同GroEL的mRNA及上下游各1000bp段基因间区存在潜在靶向结合。不同pH培养条件下的高、低致龋性变异链球菌中,sRNASpR19均低表达,GroEL的蛋白水平与RNA水平均高表达,结合生物信息学分析提示sRNA SpR19可能通过靶向GroEL负调控变链菌的致龋能力,将来两者可能作为一对标志物分子用于鉴别高低致龋菌。
[Abstract]:Caries, as a chronic, multifactorial disease, affects human health. Streptococcus mutans, as the primary dental caries-causing bacteria, has a complex pathogenic mechanism and is closely related to its acid tolerance. The virulence of Streptococcus mutans to caries varied from strain to strain, and the discovery of Streptococcus mutans was attracting more and more attention. At present, The related studies are based on differential proteomics and the acquisition of key regulatory genes by high-throughput sequencing. Selex technology mainly uses single-stranded nucleotide libraries to screen aptamer (aptamer). Aptamer, also known as "artificial antibody", which binds specifically to target substance. The recognition pattern is similar to the protein antibody, and has the advantages of no immunogenicity, accurate recognition and convenient modification, so it can be used as a new molecular probe. Cell subtractive SELEX can screen out the difference in the specific recognition of two homologous target materials such as the aptamer-H19, identified by our team for the identification of high caries bacteria has potential application value in the prediction of susceptibility to caries. Bacterial non-coding small RNA (small non-coding RNA,sRNA) plays a role in the regulation of pathogenic genes at the posttranscriptional level mainly through base pairing binding to target mRNA,. The use of high-throughput screening technology combined with bioinformatics prediction has become a research topic in recent years. The role of .sRNA in environmental tolerance, bacterial virulence and drug resistance is also the focus of our research group. By screening and studying the clinical isolates of Streptococcus mutans with high and low caries, the stable clinical isolates (strain 5 and strain 4) with high and low caries were obtained. The purpose of this experiment was to study the variation of differential sRNA in acid-tolerance reaction by using specific aptamer-H19 to catch the differential proteins between high and low caries producing strains and to study the changes of differential sRNA obtained by high-throughput technique. The first part: screening and identification of differential proteins of clinical isolates of Streptococcus mutans with high and low caries. Differential proteins were identified by SDS-PAGE electrophoresis, silver staining and mass spectrometry by aptamer-H19 combined with Pull-down technique. Candidate protein information was obtained. GroEL polyclonal antibody was prepared and polypeptide was synthesized. Western Blot and qRT-PCR were used to observe the expression level of the polyclonal antibody in different pH culture conditions. It was found that there were differences in the expression of all strains 4 and 5, and the differences were more significant under acidic conditions. The second part: screening and identification of differential sRNA isolated from clinical isolates of Streptococcus mutans with high and low caries, and identification of total RNA, isolation sRNA, of strain 45 from Streptococcus mutans. The library was constructed by high throughput screening of known differential sRNA SpR19; qRT-PCR observation. Changes of SpR19 expression in clinical isolates of Streptococcus mutans with low caries under different pH conditions. The expression of high caries bacteria was down-regulated in both normal and acidic environments, contrary to GroEL mRNA. Through bioinformatics analysis, it was found that there was a potential targeting binding between SpR19 and mRNA of GroEL and the intergenic regions of upstream and downstream 1000bp segments. Under different pH culture conditions, the protein level and RNA level of pH SpR19 were all high in Streptococcus mutans with low caries. Combined with bioinformatics analysis, it was suggested that sRNA SpR19 could negatively regulate the cariogenic ability of Streptococcus mutans by targeting GroEL. In the future, they may be used as a pair of marker molecules for the identification of high and low caries causing bacteria.
【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】：2017
【分类号】：R781.1

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