Gene Cloning of Virulence Factors of Streptococcus mutans

C血清型变形链球菌细胞表面存在一个190 KD的蛋白质，该蛋白质对变形链球菌在牙齿表面的粘附起着重要作用。将变形链球菌MT 8148（C型）190 KD蛋白抗原（PAc）的结构基因克隆到质粒载体pUC 118中。SDS-聚丙烯酰胺凝胶电泳和蛋白质印迹分析表明，E.重组质粒在大肠杆菌中表达，产生190 ~ 210 KD的多条多肽。免疫扩散分析表明，克隆的PAc具有与S.变形杆菌对克隆的pac基因进行了定位，并通过表征嵌合质粒的缺失突变体来确定其转录方向。用克隆的基因序列作为探针进行Southern印迹分析，发现在血清型c/e/f S的DNA中存在同源序列。变形杆菌，但不来自血清型d/g S。sobrinus。通过将红霉素抗性基因插入到pac基因中来构建pac缺陷型突变体。突变株的细胞表面疏水性低于亲本菌株MT 8148。

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.