Development of Anti-Dental Caries Vaccine Using Recombinant Lactic Streptococci

变形链球菌被认为是人类龋病的致病微生物。变形链球菌产生一种分子质量为190千道尔顿的细胞表面蛋白抗原(PAC)。在本研究中，我们构建了携带变形链球菌pac基因的重组乳链球菌，为研制龋齿口服疫苗奠定了基础。我们克隆了变形链球菌MT8148(血清型c)的pac基因，并测定了该基因的全序列。Pac基因由4695个碱基对组成。最初的基因产物含有1,565个氨基酸，分子量为170,773.2。为了阐明Pac的生物学功能，我们构建了几个Pac基因缺陷的变形链球菌转化子。PAC缺陷转化子的细胞疏水性明显低于亲本菌株。亲水性菌株的静息细胞附着在唾液包被的羟基磷灰石上的数量比亲本菌株少。这些发现表明，变形链球菌的细胞表面PAC参与了链球菌细胞与牙齿表面获得性透明膜的附着。变形链球菌的PAC与远缘链球菌一个210千道尔顿的蛋白抗原在中心区域有很高的同源性。将变形链球菌的pac基因连接到穿梭载体pSA3中。成功地将嵌合表达载体pSM1转化乳酸链球菌。重组乳酸链球菌产少量PAC。重组乳酸链球菌口服免疫小鼠后，可诱导唾液免疫球蛋白A和血清免疫球蛋白G对PAC产生明显的免疫应答。

Streptococcus mutans has been implicated as a causative organism of human dental caries. S. Mutans produces a cell surface protein antigen (PAc) with a molecular mass of 190 kilodaltons. In this study, we constructed a recombinant lactic streptococcus with carries th pac gene of S. Mutans for development of an oral vaccine against dental caries.1. We cloned the pac gene of S. Mutans MT8148 (serotype c) and determined the complete nucleotide sequence of the gene. The pac gene consisted of 4,695 base pairs. The initial gene product contained 1,565 amino acids and had a molecular weight of 170,773.2. To make clear the biological function of PAc, we constructed genetically several PAc-defective transformants fo S. mutans. The cell hydrophobicity of PAc-defective transformants was markedly lower than that of the parent strain. Resting cells of the hydrophilic strains attached in lower number to saliva-coated hydroxyapatite than did the parent strain. These findings suggest that cell surface PAc of S. Mutans participates in attachment of the streptococcal cell to aquired pellicles on tooth surfaces.3. Homology between PAc of S. Mutans and a 210-kilodalton protein antigen of Streptococcus sobrinus was high in the central regions.4. The pac gene from S. mutans was joined to shuttle vector pSA3. The chimeric plasmid pSM1 was successfully transformed into Streptococcus lactis. The recombinant S. Lactis produced a small amount of PAc. Significant salivary immunoglobulin A and serum immunoglobulin G responses to PAc were induced in mice immunized orally with the recombinant S. lactis.