The Role And Mechanism Of Streptococcus Mutans Biofilm In Cariogenic Process

Biofilm is a micro-ecological structure in which bacteria accumulate and adhere to tissues and surfaces,and encapsulates themselves with extracellular macromolecules for complex physiological metabolic activities.Various major biological macromolecules such as proteins,polysaccharides,DNA and the like present in extracellular polymeric substances(EPS).There is a family of proteins with high affinity for DNA,the DNA binding protein family(DNABII),which includes histones HU and some aggregate host factor proteins IHF expressed in Gram-negative bacteria,only in Streptococcus mutans.There is a histone-like HU.The DNABII protein family forms a three-dimensional structure by binding DNA cross-linking,which plays an important role m the development and progression of biofilm.Studies on bacteria such as Escherichia coli and Pseudomonas aeruginosa have shown that antiserum targeting DNABII family proteins can destroy the structure of biofilms,thereby allowing bacteria to be exposed from biofilms,and then using antibacterial drugs in combination.The effect of antibacterial treatment.We studied the Streptococcus mutans biofilm by preparing specific antibodies against HU.ObjectiveTo explore the mechanism of action of Streptococcus mutans as a biofilm in the development of dental caries.The in vitro model of Streptococcus mutans biofilm was used to analyze the adhesion,aggregation and biofilm formation and development of Streptococcus mutans.The experimental study on the environmental conditions of biofilm formation,protein factors and other related factors mediated the development of rickets caused by Streptococcus mutans.Recombinant expression of Streptococcus mutans HU protein,and preparation of HU-specific antibodies for the action of HU protein on the formation of Streptococcus mutans biofilm.Method1.Using a biofilm formation model of bacteria on a 96-well plate.Under the anaerobic conditions,Streptococcus mutans was cultured with sucrose-containing brain heart infusion medium BHI,and the in vitro biofilm structure was formed on the 96-well plate.The nucleus of the bacteria in the biofilm was stained by crystal violet staining,and the acetic acid was washed.The deionized crystal violet was read,and the amount of biofilm formation was evaluated by reading the OD value of the eluate with a microplate reader.2.Molecular cloning of the gene fragment encoding the HU protein in the whole genome of Streptococcus mutans,and setting the specific primer with restriction enzyme digestion to obtain the HU gene fragment,and constructing the PCR product with restriction endonuclease and DNA ligase.The pET-28a-HU expression vector was transformed into BL21(DE3)competent cells and the HU protein of Streptococcus mutans was recombinantly expressed by prokaryotic expression system.3.The immune serum was obtained by immunizing the white rabbit with HU protein,and then the HU polyclonal antibody was purified by the HU protein-binding resin column.At the same time,the phage antibody library technology was used to screen the monoclonal antibodies using the recombinantly expressed HU protein as an antigen.The obtained monoclonal antibody and polyclonal antibody were subjected to ELISA and western blotting to evaluate the affinity of the antibody to HU.4.The antibody was introduced into the 96-well plate Streptococcus mutans in vitro biofilm model.The biofilm formation of the treatment group and the blank control group were compared and the OD value obtained by crystal violet staining was statistically analyzed.Results1.In vitro biofilm formation of Streptococcus mutans in 96-well plates.The sucrose component in the medium is indispensable.Sucrose not only provides energy for the growth and growth of Streptococcus mutans,but also has a structural structure for biofilm formation.The importance of Streptococcus mutans biofilm formation increases with increasing sucrose concentration,whereas in the absence of sucrose,Streptococcus mutans cannot form biofilms even if additional glucose is provided in the culture.2.Successfully cloned the HU gene fragment from the whole genome of Streptococcus mutans ingbritt strain,and increased the restriction enzyme sites in the PCR 5' and 3'primer design,and obtained the HU with BamHI and Xhol cleavage sites in the upstream and downstream.The pET-28a-HU expression vector was successfully constructed by restriction endonuclease and T4 ligase,and transferred into BL21(DE3)competent cells to obtain recombinant protein engineering strain of HU protein.The results showed that under the induction of O.lmM IPTG at 37?,the engineered bacteria could successfully express HU protein,and mainly existed in the supermatant of cell sonie lysate.3.The immune serum was successfully obtained by immunizing the white rabbit with the recombinant protein HU as the antigen,and ELISA and western blotting experiments show that the purified polyclonal antibody can specifically bind to HU.The antibody screening of the phage antibody display library obtained a set of antibody light and heavy chain sequences after 5 rounds of adsorption,panning,enrichnent and infection.However,after reconstitution of the antibody light heavy chain expression vector and transfection of 293E cells,the expressed monoclonal antibodies have no affinity for HU and are false positive results.4.At the sucrose concentration of 0.5g/l,the biofilm formation of Streptococcus mutans was significantly reduced in the HU polyclonal antibody treatment group compared with the blank control group.ConclusionsIn this study,the sucrose concentration was adjusted in the in vitro biofilm model of Streptococcus mutans.The original concentration was excessive in the process of biofilm formation,and the increase and decrease of sucrose to a small extent and the influence of other factors on the rate and result of biofilm formation.May be compensated by the concentration of sucrose.The current concentration is a lower concentration,which can be more sensitive when a treatment factor is introduced.We carried out molecular cloning and recombinant expression of the HU protein of Streptococcus mutans,and obtained a polyclonal antibody that specifically binds HU.The polyclonal antibody was introduced as a treatment factor into Streptococcus mutans in a biofilm experiment with a lower sucrose concentration,and it was found that the biofilm formation of Streptococcus mutans was inhibited to some extent.