变异链球菌低pH感应系统的构建

Construction of a low-pH-sensing system in Streptococcus mutans

目的构建变异链球菌低p H感应系统,原位可视地检测其所处环境的p H。方法通过聚合酶链反应(PCR)和基因重组技术,克隆得到唾液链球菌57.I的ureⅠ基因启动子片段(pureⅠ)及绿色荧光蛋白(GFP)的编码基因gfp,经过酶切后将两个基因片段连接融合,然后再经双酶切将融合片段与大肠杆菌-变异链球菌穿梭载体p DL278连接,构建出重组质粒p DL278-pureⅠ-gfp。将重组质粒转化到变异链球菌UA159中,使用荧光显微镜观察其在不同p H条件和不同处理时间的单位面积荧光强度。结果目的基因pureⅠ和gfp扩增片段大小分别为450 bp和717 bp,与预期大小相符。构建的重组质粒p DL278-pureⅠ-gfp测序结果与数据库比对完全一致。重组变异链球菌低p H报告菌株PCR扩增片段与预期结果相符。在一定范围内,变异链球菌低p H感应系统单位面积荧光强度随p H值的降低和处理时间的延长而增强。结论本研究成功构建了变异链球菌低p H感应系统,同时验证了唾液链球菌酸诱导启动子pureⅠ能在变异链球菌中正常发挥功能,为今后研究菌斑生物膜中原位p H的动态变化提供了新方法。

Objective To construct a low-pH-sensing system in Streptococcus mutans （S. mutans） and to visually detect the pH in situ. Methods Promoter of urease I （Pure I ） and green fluorescence protein （gfp） DNA fragments were amplified by polymerase chain reaction （PCR） from the genome of Streptococcus salivarius 57.I and S. mutans containing the gfp fragment. The two amplified DNA fragments were ligated together and further integrated into pDL278 to construct the recombinant plasmid pDL278-pure I -gfp. This recombinant plasmid was then transformed into S. mutans UA159 cells. Subsequently, the intensity of the optical density per unit area of the low-pH-sensing system was measured and compared under different pH conditions and different processing times. Results Pure I and gfp DNA fragments were amplified successfully with the correct molecule sizes （450 and 717 bp, respectively）. The recombinant plasmid pDL278-pure I -gfp was constructed and fur- ther verified by PCR and sequencing. The intensity of the optical density per unit area of the low-pH-sensing system increased with decreasing pH and increasing processing time. Conclusion A low-pH-sensing system was constructed successfully in S. mutans. Our research verified that pure I of Streptococcus salivarius can function well in S. mutans as an acid induced promoter, and provided a new method of detecting the pH of plaque biofilms in situ.