| Objective：Caries disease is a teeth hard tissue disease,which has many influencing factors,butthe most important factor is bacterial infection.Streptococcus mutans play an importantrole in the development process of caries disease,and maily showed in the ability ofproducing acid and acid tolerance.With the development of modern molecular biologyand further research of the acid tolerance of Streptococcus mutans,many related acidtolerance genes have been identified and isolated，including comD and gbpB genes.Theefficacy of Fluoride to prevent caries disease has been recognized,and is widely used bythe world.Scientific experiments have proved that the use of the right amount of fluoridecan effectively prevent the happening of caries disease,however, the application of alarge amount for a long time of fluoride can lead to the growth of fluoride-resistantstrain.It has a stronger ability of cariogenicity than its parental wild type strain.Comcompetent genes family is one of the important two-component signaling transductionsystem.(TCSTS).comD gene encoding a dimerization transmembrane receptor can feelspecific stimuli out side and change it into the intracellular signaling which produceing aseries of biological character changed.gbpB has an important role in adhesion ofstreptococcus mutans, which act as“bridgeâ€between bacteria and matrix of dentalPlaque.However, the relationship between the acid-resistant genes comD and gbpBmutations of the fluoride-resistant strain of streptococcus mutans and enhancement offluoride-resistant strains resistant to acid has not been reported.In this study, we firstinduced the fluoride-resistant strain of Streptococcus mutans in vitro in order todetection of mutation in the acid-resistant genes comD and gbpB. It can be best toproclaim the cariogenicity mechanism of Streptococcus mutans from the level of geneand clearly reaveled the relationship between the acid tolerance genes offluoride-resistant strain and cariogenic strength.The study can provide a solidfoundation for rational use of fluoride and the development of new anti-caries vaccine.Methods：1) Inducing the fluoride-resistant strain of Streptococcus mutans UA159in vitro.2) Extracting the genomic of RF-UA159.3) The acid tolerance related genes comD and gbpB were PCR using genomic DNA of RF-UA159as template.4) Connecting the PCR production by pMD18-T vector, and transform it into E.coliJM109to recombine plasmid.5) Extracting the plasmid,indentifing by PCR， enzyme digestion and genesequencing.Result：We have successfully constructed the recombinant plasmid.By using the BLASTtool to compare the homology of the obtained comD and gbpB genes sequences in theGenBank datebase.We found that the acid resistance related gene gbpB of RF-UA159did not have any difference compapred to the dfp gene published in theGenBank.However, the acid resistance related gene comD of Streptococcus mutansfluoride-resistant is not as same as the comD gene published in GenBank. There is onemutation at1795538(A G).Conclusion:Successfully built the recombinant plasmids of the acid-resistant genes comD andgbpB mutations of the fluoride-resistant strain of streptococcus mutans in competent cellE.coli JM109and indentified them by PCR，enzyme digestion and gene sequencing.Theresearch is of great importance for preventing and controlling the caries disease in thefuture. |
