【作者】 刘莉；

【导师】 张志民；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2011， 硕士

【摘要】 龋病是人类最常见的细菌感染性疾病之一。变形链球菌是人类龋病的始发因素,通过对牙面的粘附、产酸及耐酸表现其致龋性,是目前公认的最重要的致龋菌。氟化物是目前最有效的防龋致剂,因此得到在全世界范围内的广泛应用。然而为了提高并维持菌斑内较高的氟化物水平,更好的预防和治疗龋病,局部应用氟化物的浓度和次数逐渐增加,将有可能导致耐氟菌株的选择性生长。耐氟菌株与亲代菌株相比具有更强的耐氟性、产酸和耐酸能力,致龋能力也随之增强。变形链球菌耐氟菌株耐酸能力的提高,是其致龋性提高的一个重要因素,对于耐酸相关基因的研究,一直是国内外学者研究的热门。目前本课题组在前期的研究中已检测出变形链球菌耐氟菌株中耐酸相关基因ffh、dgk、dltc等发生突变,突变的具体位置也已经清楚,结果已登陆美国GenBank。本实验以变形链球菌耐氟菌株耐酸相关基因dfp和dnaK为目的基因。采用体外人工诱导法诱导变形链球菌耐氟菌株,根据GenBank发表的变形链球菌耐酸相关基因dfp和dnaK的序列,根据引物设计原则,在保守区分别设计引物进行PCR,获得目的基因片段,用pMD18-T载体进行T/A连接,转入大肠杆菌感受态细胞构建重组质粒,交由生化公司进行测序鉴定。结果发现变形链球菌耐氟菌株耐酸相关基因dfp没有发生突变,而dnaK与GenBank报告序列不同,有3个碱基发生突变。突变发生的位点分别在513(T→A),1029(T→A),1172(T→C)。更多还原

【Abstract】 Caries is a common illnesses harm to human health. It has been demonstrated that 70-100% of pathogen found in human dental caries is Streptococcus mutans and Streptococcus mutans is widely recognized as the originating factor of human caries as well as the most important cariogenic bacteria. Streptococcus mutans is a Gram-positive bacteria and it can produce acid in the process of metabolism of carbohydrates from food after attaching to the tooth surface which is in accordance with that acid tolerance is the main character of streptococcus mutans found in caries.Acid resistance, as a important cariogenic factor, has attracted much attention from scholars. Acid resistance, which is defined as that bacteria can grow in the acidic environment and has the ability of producing acid in the process of metabolism of carbohydrates from food, includes constitutive acid tolerance and acid tolerance response. Acid tolerance of Streptococcus mutans is determined by a variety of factors. With deeper research of acid resistance of Streptococcus mutans, genes related to acid resistance of Streptococcus mutans has been isolated and cloned by the application of modern molecular biology techniques.Fluoride as a common toothpaste additive is used widely to prevent dental caries effectively, it can inhibit glucosyltransferase’s activity and reduce yield of ATP as well as the generation of acidic metabolic end products, which increase with the elevated concentration gradually. But the long-term application of fluoride can cause a wide range of mutation in Streptococcus mutans, resulting in the fluoride-resistant strains of Streptococcus mutans. Compared with their parent strains, fluoride-resistant strains of Streptococcus mutans can survive higher level fluoride in environment and show stronger fluoride tolerance. In the study of fluoride-resistant strains Streptococcus mutans, it has been found that capability of fluoride-resistant strains of Streptococcus mutans to cause caries is enhanced compared with the parent strains, including the ability to produce acid and acid resistance and changed genotype, but the exact location of gene mutations is not clear and requires further study. In this study, fluoride-resistant strains of Streptococcus mutans was first artificially induced in vitro and its genome was extracted by application of V-gene morsel bacterial genomic DNA kit and a pair of dfp primers and two pairs of dnaK primers were designed from the most conservative region according to different strains of Streptococcus mutans acid tolerance related gene dnaK and dfp sequences in GenBank, then genome of Streptococcus mutans fluoride-resistant strains was used to be templates for PCR to obtain acid-resistant strains of Streptococcus mutans fluoride-resistant gene dfp. dnaKl and dnaK2 gene fragments. After recovery of PCR products by kit and T/A connection by pMD18-T vector, the product was transformed into E.coli competent cells (E.coli JM109) to recombine plasmids and the plasmids was extracted and digested and identified by PCR, sequenced recombinant plasmid DNA at last. The sequenced recombinant plasmid was connected to obtain fluoride-resistant strains of Streptococcus mutans acid resistant genes dfp and dnaK. After homology comparison of the obtained sequence by using BLAST tool in the GenBank database, we found acid resistance related gene dfp of strand of Streptococcus mutans fluoride-resistant is the same as the dfp gene published in GenBank. It shows that the gene did not have any mutations. However, acid resistance related gene dnaK of strand of Streptococcus mutans fluoride-resistant is different from the dnaK gene published in GenBank. There are three mutations at 513(T→G) and 1029(T→A) and 1172(T→C).The study on acid resistance related gene dfp and dnaK of strand of Streptococcus mutans fluoride-resistant laid foundation for more reasonable application of fluoride to prevent dental caries, and it is very meaningful to construct suitable antigens for anti-caries vaccine to aquire active immunization.更多还原