Gene Cloning of Virulence Factors of Streptococcus mutans

血清型c型变形链球菌细胞表面有一种190kd的蛋白，它对细菌粘附在牙齿表面起重要作用。将S.mutans MT8148 （c型）190kd蛋白抗原（PAc）结构基因克隆到质粒载体pUC118中。sds -聚丙烯酰胺凝胶电泳和western blotting结果表明，携带嵌合质粒的大肠杆菌产生了190 ~ 210 KD的多肽。免疫扩散分析显示，克隆的PAc具有与S. mutans PAc相同的特异性决定因素。克隆的PAc基因被定位，并通过嵌合质粒的缺失突变体特征确定其转录取向。以克隆的基因序列为探针进行Southern blot分析，发现c/e/f型S. mutans的dna中存在同源序列，而d/g S. sobrinus的dna中不存在同源序列。通过在pac基因中插入红霉素抗性基因，构建了pac缺陷突变体。突变体的细胞表面疏水性低于亲本菌株MT8148。

A 190 KD protein located on the cell surface of serotype c streptococcus mutans plays an important role for adherence of the organism to the tooth surface. The structural gene for the 190 KD protein antigen (PAc) of S.mutans MT8148 (type c) was cloned into the plasmid vector pUC118. SDS-polyacrylamide gel electrophoresis and western blotting showed that the E. coli harboring the chimeric plasmid produced multiple polypeptides of 190 to 210 KD. Immunodiffusion analysis revealed that the cloned PAc had the same specific determinants as the S. mutans PAc. The cloned pac gene was mapped, and it transcriptional orientation was determined by characterizing deletion mutants of the chimeric plasmid. Southern blot analysis with the cloned gene sequence as a probe revealed the presence of a homologous sequence in DNAs from serotypes c/e/f S. mutans, but not from serotypes d/g S. sobrinus. PAc-defective mutants were constructed by the insertion of an erythromycin-resistance gene into the pac gene. Cell surface hydrophobicity of the mutants was lower than that of the parent strain MT8148.