DENTAL PLAQUE REDUCTION BY GENETIC ENGINEERING

通过基因工程减少牙菌斑

• 批准号: 3221522
• 负责人: Robert G Quivey
• 金额: $ 14.75万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3221522
• 关键词: Escherichia coli; Moniliales; SDS polyacrylamide gel electrophoresis; Streptococcus mutans; Streptococcus sanguis; biological products; biotechnology; cell transformation; chromosome translocation; complementary DNA; dental caries; dental plaque; disease /disorder model; enzyme mechanism; genetic manipulation; genetic promoter element; genetic terminator element; glucan 1,4 alpha glucosidase; laboratory rat; microorganism genetics; microorganism growth; molecular cloning; oral bacteria; plasmids; polymerase chain reaction; preventive dentistry; protein sequence; protein signal sequence; recombinant DNA; southern blotting; tooth surface; western blottings

Dental plaque constitutes an ecological niche, activities of which are highly correlated with the onset and development of dental caries. Insoluble glucan, termed mutan, provides the molecular framework around which plaque accumulates. The goal of the project is to reduce the bulk of plaque by disrupting its molecular architecture. We will then determine the effect of that disruption on the prevalence of dental caries. In vitro studies have demonstrated that applications of mutanase have substantially reduced the accumulation of plaque carbohydrate. Results from studies conducted in animals and humans have repeatedly shown that topical applications of mutanase [alpha(1->3) glucan, 3-glucanohydrolase] reduce plaque formation and caries. Thus, mutanase may have promise in the prevention of dental caries. The results from studies conducted on humans showed that mutanase, presented in chewing gum, reduced the amount of plaque and gingivitis compared with placebo groups. However, in the oral environment, the enzyme had to be applied to the tooth surface too frequently to be practical. We propose a mechanism to continuously supply mutanase to the location where it would be most efficacious, that is, within dental plaque. Cloning of the mutanase gene into Streptococcus sanguis, an organism that normally colonizes the tooth surface, represents such a mechanism. In addition, the achievement of this goal would clarify the role of mutan in the formation of plaque and the pathogenesis of oral disease. Recombinant DNA techniques will be used to clone the gene for mutanase from the fungus Trichoderma harzianum. The gene will be subcloned into a novel streptococcal expression-secretion vector and introduced into S. sanguis. The genetic construction used will permit the recombinant organism to synthesize and secrete a catalytically active enzyme (mutanase). The recombinant S. sanguis will be used in studies, together with S. mutans, in an in vitro model to assess its effect on plaque accumulation and then in the rat model system to determine its effectiveness against the development of caries. These studies will result in: a) an enhanced understanding of the role of dental plaque in the pathogenesis of dental enhanced understanding of the role of dental plaque in the pathogenesis of dental caries and information that will aid in the prevention of caries; b) an understanding of streptococcal promoters and signal sequences required for secretion of foreign gene products; c) an increased knowledge of heterologous DNA stability in the oral streptococci; and d) a novel method for the prevention of caries may be developed. The completion of this project will establish important new principles for the application of molecular biology to the prevention and treatment of oral disease.

牙菌斑构成了一个生态位，其活动是 与龋齿的发生、发展密切相关。 不溶性葡聚糖，称为“mutan”，提供了周围的分子框架 斑块积聚。 该项目的目标是减少大量 通过破坏其分子结构来形成斑块。 然后我们将确定 这种破坏对龋齿患病率的影响。 体外 研究表明，mutanase 的应用极大地促进了 减少斑块碳水化合物的积累。 研究结果 在动物和人类身上进行的研究多次表明，局部 mutanase [α(1->3) 葡聚糖、3-葡聚糖水解酶] 的应用减少 菌斑形成和龋齿。 因此，mutanase 可能在以下方面有希望： 预防龋齿。 对人类进行的研究结果 研究表明，口香糖中存在的齿龈酶可减少 与安慰剂组相比，牙菌斑和牙龈炎。 然而，在口语中 环境，酶也必须涂在牙齿表面 经常要实用。 我们提出了一种持续供应的机制 将突变酶转移到最有效的位置，即 牙菌斑内。 将变异酶基因克隆到链球菌中 血杆菌是一种通常定植于牙齿表面的生物体，代表 这样的机制。 此外，这一目标的实现将澄清 mutan 在牙菌斑形成和口腔溃疡发病机制中的作用 疾病。 重组DNA技术将用于克隆该基因 来自真菌哈茨木霉的变异酶。 该基因将被亚克隆 入新型链球菌表达分泌载体并引入 血红沙门氏菌。 所使用的基因构建将允许重组 生物体合成和分泌具有催化活性的酶 （变异酶）。 重组血链球菌将用于研究，一起 与变形链球菌一起在体外模型中评估其对斑块的影响 积累，然后在大鼠模型系统中测定其 有效对抗龋齿的发展。 这些研究将产生 a) 加深对牙菌斑在牙周病中的作用的了解 牙齿发病机制 增强对牙菌斑作用的认识 龋齿的发病机制以及有助于治疗的信息 预防龋齿； b) 了解链球菌启动子和 分泌外源基因产物所需的信号序列；能 增加对口腔链球菌异源 DNA 稳定性的了解； d) 可以开发一种预防龋齿的新方法。 这 该项目的完成将为 分子生物学在口腔疾病预防和治疗中的应用 疾病。