【作者】 朱来宽；

【导师】 张志民；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2007， 硕士

【摘要】 龋病是牙体硬组织的细菌感染性疾病,变形链球菌是其主要的致病菌,耐酸性是变形链球菌致龋性的一个重要方面,耐酸基因的研究受到国内外学者的密切关注,目前在一些原核生物中已分离、测序并克隆了多个耐酸性相关基因。在对变形链球菌耐氟菌株的研究中发现,其耐酸性增强,并且基因型发生改变,但基因突变的具体部位目前尚不清楚。本实验首先在体外人工诱导变形链球菌耐氟菌株(国际参考株S. mutans ingbritt血清型c),用V-gene细菌基因组DNA小量制备试剂盒提取细菌基因组,根据GenBank发表的变形链球菌fhs基因序列,设计引物进行PCR,获得变形链球菌耐氟菌株耐酸基因fhs的部分基因序列,利用PGEM-T载体进行T/A克隆,构建重组质粒,并进行测序鉴定。结果在变形链球菌耐氟菌株中发现其耐酸相关基因fhs发生突变,并且变形链球菌耐氟菌株耐酸相关基因fhs已登陆美国GenBank,序列号为EF405868。更多还原

【Abstract】 Caries is dentine disease infected by bacteria. Streptococcus mutans is recognized main cariogenic bacteria.Fluoride is extensively used for caries prevention. Its effect on cariogenic bacteria is one of important mechanisms for the caries prevention by fluoride. But long-term topical applications of high-concentration fluoride may lead to growth of F-resistant strains. F-resistant strains depress the effect of topical applications of fluoride and are more difficult to be restrained than the parent strains. The appearance of F-resistant strains bring the new issue for caries prevention by fluoride. Studying the F-resistant strains can make us know the changes of cariogenicity of S. mutans after fluoride - resistant mutation beforehand, afford experimental evidence for using fluoride rationally and finding new medication.Streptococcus mutans is recognized as cariogenic bacterium whose cariogenicity is related with acidogenic ability and acid resistance. It is reported that the acidogenic ability and acid resistance of S. mutans increased after S. mutans fluoride - resistant mutated. Acid resistance is that bacteria can continue producing acid and performing its function under low pH value,which includes constitutive acid tolerance and acid tolerance response. Its acid resistance is decided by many genetic factors. Some acid-resistant genes of S. mutans have been separated and cloned now. In this report we study the gene: fhsThe gene encodes formyl-tetrahydrofolate synthetase (Fhs) from both prokaryotic and eukaryotic sources. In many bacteria, Fhs catalyzes the formation of 10-formyl-tetrahydrofolate, which is used directly in purine biosynthesis and formylation of Met-tRNA and indirectly in the biosynthesis of methionine, serine, glycine, and thymine. For ATP is the substrate of H+-ATPase affection,we predicted that themutation induced the abnormal of acid resistance which because the purine synthesis abnormal cause ATP synthesis abnormal,which induced H+-ATPase remove H+ abnormal.It is repored that The genotypes of F-resitant strains of S. mutans have been changed and may comprise several gene mutations,but idiographic part of gene mutation is not known. In this study, we designed the primers according to fhs’s sequence of S. mutans from GenBank database to do PCR. So we got the fhs’s partial sequence of the F-resitant strains. We made the acquired sequences connected with PGEM-T vector and sequenced them. We compared the sequence with it from GenBank database by blastn and found that fhs’s sequence has 6 bases to be changed. The results were submitted to American GenBank, and the accession number is EF405868. Fhs’s mutation is point mutation and the substitution cause two changes in the amino acid sequence of the protein. These changes are Arg→Gln and Val→Ile. We can guess that the mutations of acid-resistant gene ffh relate to the advance of the acid-resistant characteristic of the fluoride-resistant strain of streptococcus mutans.As is extensively used for preventing caries,fluoride is challenged by F-resistant strains.So we have to find new medicine for caries preventive,fluoride and we should use fluoride in right way.The mechanism of fluorid to prevent caries is that it can effect both teeth and bacteria.It is reported vaccine for streptococcus mutans can prevent caries.With the development of the technology of molecular organism,we have got great achievement on vaccine. DNA vaccine is the great progress in human preventive medicine. It’s theory is making exogenous gene imported into host cell, inducing host immune system to carry out immune reaction. The study on acid-resistant gene fhs prefers new direction for DNA vaccine and has important significance.更多还原