DENTAL PLAQUE REDUCTION BY GENETIC ENGINEERING

通过基因工程减少牙菌斑

基本信息

批准号：
2129895
负责人：
Robert G Quivey
金额：
$ 15.57万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-09-01 至 1995-08-31
项目状态：
已结题

项目摘要

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.

牙菌斑构成生态位，其活动是与龋齿的发作和发展高度相关。不溶性葡萄糖，称为utan，在周围提供了分子框架哪个斑块积聚。该项目的目的是减少大部分通过破坏其分子建筑的牌匾。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，肌酶的应用大大具有减少了斑块碳水化合物的积累。研究结果在动物和人类中进行的一再表明局部胰蛋白酶的应用[α（1-> 3）葡聚糖，3-葡萄糖液酶]降低斑块形成和龋齿。因此，突变酶可能有望预防龋齿。对人类进行的研究的结果表明在咀嚼口香糖中呈现的突变酶减少了与安慰剂组相比，斑块和牙龈炎。但是，在口头环境，酶也必须应用于牙齿表面经常是实用的。我们提出了一种连续供应的机制杂种酶到最有效的位置，即在牙菌斑内。将突变酶基因克隆到链球菌中 Sanguis是一种通常在牙齿表面殖民的生物，代表这样的机制。此外，这个目标的成就将澄清 Mutan在牙菌斑形成和口服发病机理中的作用疾病。重组DNA技术将用于克隆基因真菌trichoderma harzianum的mutanase。该基因将被亚克隆进入新型链球菌表达 - 分泌载体，并引入 S. Sanguis。所使用的遗传构建将允许重组有机体合成和分泌催化活性酶（mutanase）。重组S. sanguis将共同用于研究使用S. mutans，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确定其反对龋齿发展的有效性。这些研究将导致在：a）对牙齿在牙齿的发病机理增强对牙齿作用的理解在龋齿和信息的发病机理中，将有助于预防龋齿； b）了解链球菌启动子和外国基因产物分泌所需的信号序列；能口服链球菌中异源DNA稳定性的知识增加； d）可以开发一种预防龋齿的新方法。这该项目的完成将建立重要的新原则分子生物学应用于预防和治疗口服疾病。