Development of immunological method for prevention of dental caries using genetic engineering

变形链球菌诱导龋齿的第一步是生物体粘附到牙齿表面。表面蛋白抗原（PAc）被认为参与了细胞在牙齿表面获得性膜上的非蔗糖依赖性附着。另一方面，葡糖基转移酶（glucosyltransferase，GTF）在变形链球菌的蔗糖依赖性粘附中起重要作用，我们利用多种十肽进行抗原表位定位，发现抗原表位位于PAc的N端A重复序列和中间区域，并且A重复序列与唾液组分结合。因此，我们使用表达质粒载体pTrc 99 A构建了对应于PAc的A重复序列的片段和对应于GTF-1的蔗糖结合位点的片段的融合蛋白。含有嵌合质粒pFPG 22的大肠杆菌XL 1-blue表达了PAc和GTF-1的融合蛋白，其与兔抗PAc血清和抗GTF-1血清均反应。用该融合蛋白皮下免疫诱导高滴度的抗体的粘附因子，这些抗体抑制GTF-I，这表明融合蛋白可能是有用的免疫预防龋齿。

The first step for induction of dental caries by Streptococcus mutans is the adherence of the organism to tooth surfaces. Surface protein antigen (PAc) is considered to participatein the sucrose-independent attachment of the cell to acquired pellicles on tooth surfaces. On the other hand, glucosyltransferase (GTF) is important in the sucrose-dependent adherence of S.mutans.Our epitope mapping using many decapeptides showed that antigenic epitopes were located in the N-terminal A-repeat and middle region of PAc.Moreover, the A-repeat bound to salivary components. We have, therefore, constructed a fusion protein of the fragment corresponding to the A-repeat of PAc and the fragment corresponding to the sucrose-binding site of GTF-I,using an expression plasmid vector pTrc99A.Escherichia coli XL1-blue containing the chimeric plasmid pFPG22 expressed a fusion protein of PAc and GTF-I that reacted with both rabbit anti-PAc serum and antiGTF-I serum. Subcutaneous immunization with this fusion protein induced high titers of antibodies to both adherence factors, and these antibodies inhibited GTF-I, suggesting that the fusion protein may be useful for immunological prevention of dental caries.