变形链球菌PrA蛋白在致龋过程中新的生物学功能研究

Streptococcus mutans are the uppermost cariegenesic bacteria in human. The streptococcus mutans caries factors is the critical factor in the process of dental caries occurrence. But, the effect of the dental caries prevention and treatment based on the single streptococcus mutans caries factors is not satisfactory at present. Whether there are now unknown factors to cooperate with or adjust to the role of streptococcus mutans caries factors in the process of dental caries occurrence? According to this thought, we put streptococcus mutans as a whole, and screen specific aptamers of the high cariegenesic Streptococcus mutans strains isolated from clinical individuals using the subtractive SELEX techniques targeted the whole cells established by us. Subsidized by the national natural science foundation of china (NSFC), we have get the specific DNA aptamers of the high cariegenesic Streptococcus mutans strains isolated from clinical individuals and these aptamers can inhibit the activity of adhesion, the different proteins were tagged by targeted the screened different ssDNA aptamers and mass spectrometry analysis showed that the different proteins are GTF and the unknown function protein PrA now. In this study, we clone and express PrA, and research the binding activity between PrA and different aptamers, research the effect of PrA in the process of dental caries occurrence and the interaction relationship between GTF and PrA by gene knockout, on the basis of PrA was tagged successfully in the past. Through the studies in the project, we hope to explicit the biological function of PrA in the process of dental caries occurrence, and provide new ideas for the caries research as well as new scheme for clinical caries prevention and treatment.

变形链球菌是人类口腔中最主要的致龋菌，致龋因子是变形链球菌致龋的关键因素，但目前针对单一致龋因子的龋病防治效果还不能令人满意。是否还存在目前我们未知的因素在变形链球菌致龋过程中协同或调节致龋因子的致龋作用？按照这一思路，我们把变形链球菌作为一个整体，利用我们建立的完整细胞消减SELEX技术，筛选高致龋性变形链球菌特异配基，在前一个国家自然科学基金的资助下，我们已经筛选获得特异识别高致龋性变形链球菌并且具有抑制粘附活性的DNA配基，利用DNA配基钓取了差异蛋白，质谱分析显示差异蛋白为GTF和目前功能未知的蛋白PrA。本研究在以往我们成功钓取PrA的基础上，克隆、表达PrA，研究PrA与差异配基的结合活性，通过基因敲除研究PrA在变形链球菌致龋过程中的作用，以及PrA与GTF的相互作用关系。通过本项目研究希望明确PrA在致龋过程中的作用，为龋病防治研究提供新的思路，为临床龋病预防提供新的方案。

变形链球菌是人类口腔中最主要的致龋菌，我们已经筛选获得特异识别高致龋性变形链球菌并且具有抑制粘附活性的DNA配基，利用DNA配基钓取了目前功能未知的差异蛋白PrA。本研究首先克隆、表达和纯化了PrA；凝胶阻滞试验证实了PrA蛋白与H19 配基特异性结合；构建了变形链球菌PrA和GTF缺陷菌株及PrA和GTF双缺陷菌株，并进行了形态和分子生物学鉴定；完成了缺陷株致龋能力的研究工作，证实PrA和GTF缺陷菌株及PrA和GTF双缺陷菌株的致龋能力较野生菌株弱；建立了动物实验研究模型，并根据建立的动物实验模型，完成了缺陷株动物实验研究工作，相关动物实验研究结果正在分析整理中。通过本项目研究希望明确PrA在致龋过程中的作用，为龋病防治研究提供新的思路，为临床龋病预防提供新的方案。