Development of Immunological Procedure for Prevention of Dental Caries Using Fusion Protein of Colonization Factors

变形链球菌产生的细胞表面蛋白抗原(PAC)和葡萄糖转移酶(GTF)被认为是生物体的主要定植因子，抑制这两种因子有望预防龋病。本研究构建了PAC的唾液结合富含丙氨酸区域(PACA)和GTF-I的葡聚糖结合(GB)结构域的融合蛋白PACA：GB和PACA的融合蛋白PACA：SB和GTF-I的蔗糖结合(SB)结构域，并从含有融合基因的大肠杆菌细胞提取液中纯化了重组融合蛋白，并制备了兔抗体。抗PACA：GB抗体对变形链球菌细胞结合和无细胞GTF合成水不溶性葡聚糖和GTF-I合成总葡聚糖有明显的抑制作用，而抗PACA：SB抗体对其无明显抑制作用。当在反应混合物中加入抗PACA：GB抗体时，变形链球菌对唾液包被的羟基磷灰石小球的蔗糖非依赖性和蔗糖依赖性的黏附被显著抑制。抗PACA：SB抗体在没有蔗糖的情况下抑制变形链球菌与珠子的黏附，但在蔗糖存在的情况下不抑制。用融合蛋白PACA：GB免疫可能有助于控制有机体对牙齿的定植。

Cell-surface protein antigen (PAc) and glucosyltransferases (GTFs) produced by Streptococcus mutans are considered to be major colonization factors of the organism, and the inhibition of these two factors is predicted to provide protection against dental caries. In this study, we have constructed a fusion protein PAcA : GB of the salivabinding alanine-rich region (PAcA) of PAc and the glucan-binding (GB) domain of GTF-I,an enzyme catalyzing the synthesis of water-insoluble glucan from sucrose, and a fusion protein PAcA : SB of PAcA and the sucrose-binding (SB) domain of GTF-I.The recombinant fusion proteins were purified from cell extracts of Escherichia coli harboring the fusion genes, and rabbit antibodies against these fusion proteins were prepared. The water-insoluble glucan synthesis by cell-associated and cell-free GTF preparations from S.mutans and the total glucan synthesis by GTF-I was markedly inhibited by anti-PAcA : GB IgG antibodies, but not by anti-PAcA : SB IgG antibodies. Significant inhibition of the sucrose-independent and sucrose-dependent adhesion of S.mutans to saliva-coated hydroxyapatite beads was observed when anti-PAcA : GB antibodies were added to the reaction mixture. Anti-PAcA : SB antibodies inhibited the adhesion of S.mutans to the beads in the absence of sucrose, but did not in the presence of sucrose. Immunization with the fusion protein PAcA : GB may be useful for controlling the colonization of teeth by the organism.