高、低致齲性變異鏈球菌臨床分離株關(guān)鍵差異蛋白與差異sRNA的鑒定
發(fā)布時(shí)間：2018-09-01 16:40
【摘要】：齲病,作為一種慢性、多因素疾病影響著人類健康。變異鏈球菌作為口腔內(nèi)首要致齲菌,致病機(jī)制復(fù)雜,與其耐酸性緊密相關(guān)。不同菌株間的變異鏈球菌致齲毒力可存在很大差異,這一發(fā)現(xiàn)逐漸引起關(guān)注。目前,相關(guān)的研究主要建立在差異蛋白質(zhì)組學(xué)和通過(guò)高通量測(cè)序獲取關(guān)鍵調(diào)控基因基礎(chǔ)上。SELEX技術(shù)主要利用單鏈核苷酸文庫(kù)篩選與靶物質(zhì)特異性結(jié)合的核酸適配體(aptamer)。Aptamer又稱“人工抗體”,識(shí)別模式與蛋白抗體相似,具有無(wú)免疫原性、識(shí)別精準(zhǔn)、修飾方便等優(yōu)勢(shì),作為新型分子探針應(yīng)用。細(xì)胞消減SELEX可篩選出特異識(shí)別兩種同源靶物質(zhì)上的差異aptamer,如本課題組前期獲得的識(shí)別高齲菌的aptamer-H19,在齲病易感性預(yù)測(cè)方面存在潛在應(yīng)用價(jià)值。細(xì)菌非編碼小RNA (small non-coding RNA,sRNA)則主要通過(guò)堿基配對(duì)結(jié)合靶mRNA,從而在轉(zhuǎn)錄后水平發(fā)揮調(diào)控致病基因的作用。采用高通量篩選技術(shù)結(jié)合生物信息學(xué)預(yù)測(cè)的方法成為近年來(lái)研究、獲取sRNA信息的主流。sRNA在環(huán)境耐受、細(xì)菌毒力、耐藥性等方面的作用也是本課題組的研究熱點(diǎn)。課題組通過(guò)前期對(duì)高、低致齲性變異鏈球菌臨床分離株的篩選和研究,獲得了性質(zhì)穩(wěn)定的分別具有高、低致齲特性的變異鏈球菌臨床分離株(菌株5和菌株4)。本實(shí)驗(yàn)?zāi)康氖抢锰禺恆ptamer-H19釣取高、低致齲菌株間的差異蛋白質(zhì)同時(shí)研究采用高通量技術(shù)得到的差異sRNA在耐酸反應(yīng)中的變化情況。第一部分:高、低致齲性變異鏈球菌臨床分離株差異蛋白的篩選和鑒定利用特異識(shí)別高致齲菌株的aptamer-H19結(jié)合Pull-down技術(shù)從細(xì)菌總蛋白中釣取差異蛋白,經(jīng)SDS-PAGE電泳、銀染、質(zhì)譜鑒定,獲得候選蛋白信息。制備GroEL多克隆抗體并合成多肽,采用Western Blot以及qRT-PCR觀察在不同pH培養(yǎng)條件下菌株4、5中的表達(dá)水平。發(fā)現(xiàn)其中菌株4、5皆存在表達(dá)差異且酸性條件下差異更加顯著。第二部分:高、低致齲性變異鏈球菌臨床分離株差異sRNA的篩選和鑒定提取菌株4、5總RNA,分離sRNA、構(gòu)建文庫(kù),高通量篩選出已知的差異sRNA SpR19; qRT-PCR觀察高、低致齲性變異鏈球菌臨床分離株在不同pH情況下SpR19的表達(dá)變化。無(wú)論正常還是酸性環(huán)境下高致齲菌表達(dá)量都下調(diào),與GroEL mRNA表現(xiàn)相反。經(jīng)過(guò)生物信息學(xué)分析,發(fā)現(xiàn)SpR19同GroEL的mRNA及上下游各1000bp段基因間區(qū)存在潛在靶向結(jié)合。不同pH培養(yǎng)條件下的高、低致齲性變異鏈球菌中,sRNASpR19均低表達(dá),GroEL的蛋白水平與RNA水平均高表達(dá),結(jié)合生物信息學(xué)分析提示sRNA SpR19可能通過(guò)靶向GroEL負(fù)調(diào)控變鏈菌的致齲能力,將來(lái)兩者可能作為一對(duì)標(biāo)志物分子用于鑒別高低致齲菌。
[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.
【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】：2017
【分類號(hào)】：R781.1
本文編號(hào)：2217768
[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.
【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】：2017
【分類號(hào)】：R781.1
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