| Streptococcus mutans, which is the predominant pathogen of caries, can close adhere to the surface of teeth , continue producing organic acid and resist to lower pH. Lactic acid production by fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase (LDH)is shown to be an important virulence factor forStreptococcus mutans. Caries of teeth are directly due to activities of lactate dehydrogenase, which ferment pyruvic acid into lactic acid and decrease the pH within local plaque. Under growth conditions where intracellular fructose-1,6-diphosphate concentration is high , such as in the presence of excess glucose, 90% of the glucose can be transformed into Lactic acid; but with limited glucose, fementation production other than lactate have been observed. The activity of pyruvate formate-lyase and pyruvate dehydrogenase accounts for the observed production of formate, acetate, ethanol and acetoin. In 1978, Hillman isolated one of the mutants of Streptococcus mutans which lack the enzyme activity of dehydrogenase. The mutants produced less titratable acid from glucose, adhered better to hydroxyapatite, and accumulated more plaque when grown in the presence of sucrose than did the parent strain. He proposed that the mutants might be used as an effector strain in the replacement therapy of dental caries. Then many efforts on establishing and screening effector strains were taken, but because the limited methods of chemistry and ultraviolet radiation in the early studies, the competitive ability and stability of the mutants were not ideal. With the development of the molecular biology, investigations were taken to mutate ldh gene. Following some unsuccessful attempt. the investigator demonstrate that entirely inactive ldh gene was always followed by cell death, which might because the accumulation of toxic metabolic production. In an attempt to overcome the toxic effects, the Z. mobilis adh gene was chosen as the substitution of ldh gene. At the same time, many attentions were taken on a S. mutans strain derived from clinic, which had a wide antibiotic spectrum and could inhibit other strains' ability of adherence and growth associated with the quantity of mutancin. All the properties made it the candidate of effector strain of S. mutans for replacement therapy of dental caries. An successful method is deleting virtually all of the ldh open reading(ORF) frame and introduced a supplemental alcohol dehydrogenase activity by substituting the adhB ORF from Z. mobilis, which was transformed into S.mutans and integrated with chromosome by spontaneous self- recombination. It not only overcome the lethal effect of LDH deficiency but also ensure the irreversibility of the mutation. Credible orientation of recombination avoid of various uncertain changing by affecting other genes. The resulting clone was found to be less cariogenic, stable transmissibility and non-side effect in vitro and animal experiments. We improved the technique based on the investigations mentioned before to clone and sequence the gene encode the lactate dehydrogenase and its homologous fragment of Streptococcus mutans. We did it as follows: 1,cultivation of S.mutans: Mitis Salivarious agar was used to culture S.mutans Ingbritt at the congdition of 37℃ and anaerobic cultivation for 48 h, then the strains were transmitted to liquid M-S medium and cultured for another 48h.at the same condition. 2,picking up the chromosome DNA:1.5ml culture of bacteria was centrifuged, digested by lysozyme and proteinase K, extracted by saturated hydroxybenzene and chloroform: amylene alcohol(24:1), deposited by NaAC and absolute ethanal, washed by 70% ethanal, soluted in TE and stored at-20℃. 3,PCR amplified the ldh gene and its homologous fragment of S. mutans: The reaction system of PCR was 50μl, the chromosome DNA was 1μl, Ex Taq (5U/μl) 0.5μl, 10×Ex Taq buffer(Mg2+ plus)5μl, dNTPs Mixture 4μl, 94℃3 min, circulated 30 times (94℃30s,55℃30s,72℃1min),72℃10min. then observed by 1% agarose gels electrophoresis. 4,... |
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