The Identification Of The Key Different Protein And SRNA From The High Cariogenic And Low Cariogenic Clinic Isolate Streptococcus Mutans Strain

Dental caries, as a chronic and multifactorial diseases affects the human health.Streptococcus mutans was recognized as one of the main cariogenic bacteria in the mouth,with complicated mechanism lead to caries, especially related to the ability of acidic resistance ability. Different strains of Streptococcus mutant's caries virulence can vary greatly, which is gradually attracting attention. At present, related studies are mainly focused on differential proteomics and high-throughput sequencing to obtain key regulatory genes.SELEX technology utilizes single-stranded nucleotide libraries to screen for aptamer that specifically binds to the target aptamer, also known as "artificial antibody", is similar to protein antibody, with the advantages of no immunogenicity, accurate targeting and easy to modification, which is used as a new molecular probe. Cell subtractive SELEX can screen out the the difference aptamer differences in the identification of two homologous target substances on, such as the early identification of this group of aptamer-H19, in the predisposing potential of dental caries potential application value.Small non-coding RNA (sRNA) binds to target mRNA by base pairing, and participate in regulating the pathogenic genes post-transcriptionally. The application of high-throughput screening technology combined with bioinformatics prediction method has become the mainstream of research in recent years to obtain sRNA information. SRNA relating to environmental tolerance, bacterial virulence, drug resistance and other aspects of function are also our research hotspot.Streptococcus mutans clinical isolates with high cariogenic (clinic isolate strain 5) and low cariogenic (clinic isolate strain 4) were screened by our group's previous studies. The purpose of this study is to use the specific aptamer-H19 to capture the differential proteins between high and low caries strains and to study the difference sRNA changes in the acid resistance procedure, which was acquired by high-throughput technique.Part I: Screening and Identification of Differential Proteins between High and Low Cariogenic Clinical Isolates Streptococcus Mutan strainThe differential protein was acquired from the total bacterial protein by aptamer-H19 and pull-down technique, which specifically identified the highly caries strain. By using SDS-PAGE electrophoresis, silver staining and mass spectrometry to obtain the candidate protein information. Preparation of GroEL polyclonal antibodies and synthesis polypeptides, Western Blot and qRT-PCR were used to observe the expression levels in strains 4?5 under different pH culture conditions. It was found that the strains 4 and 5 were different in expression and the difference was more significant under acidic conditions.Part II: Screening and Identification of Differentially Expressed sRNAs in Clinical Isolates of High and Low Carcinogenic Streptococcus mutansTotal RNAs from high cariogenic(clinic isolate strain 5)and low cariogenic(clinic isolate strain 4) strains were extracted ? separated, construct the library, subjected to high-throughput sequencing respectively for the profiling of differentially expressed sRNA;The differential expression level of candidate sRNA SpR19 in different cariogenic strains under different pH culture conditions was verified by qRT-PCR.The expression of high cariogenic bacteria was down-regulated both in normal and acidic environments, and GroEL mRNA performance is the opposite. After bioinformatics analysis, it was found that SpR19 had potential binding ability to the gene of GroEL and the intergenic region of 1000bp segment upstream and downstream.The expression of sRNA SpR19 was low, and the protein level and mRNA level of GroEL were higher both in high and low cariogenic Streptococcus mutans under different pH culture conditions. The application of bioinformatics analysis suggested that sRNA SpR19 could be regulated by target Streptococcus mutans cariogenic ability, in the future may be used as a pair of markers for the identification of high and low cariogenic bacteria.