Saliva-binding region of Streptococcus mutans surface protein antigen
Saliva-binding region of Streptococcus mutans surface protein antigen
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变形链球菌表面蛋白抗原的唾液结合区
作者：
M. Nakai;N. Okahashi;H. Ohta;T. Koga
变形链球菌190 kDa表面蛋白抗原(PAC)与人唾液成分结合。为了检测PAC蛋白与人唾液成分的特异性结合，使用了一种简单的夹心法。将预涂有重组PAC(RPAC)、PAC片段或变形链球菌全细胞的微滴定板与人全唾液反应，然后与生物素化的RPAC孵育。用碱性磷酸酶标记的链霉亲和素和对硝基苯磷酸检测与唾液成分结合的生物素化RPAC。在本实验中，证实了变形链球菌和纯化的RPAC的全细胞与唾液成分的结合。为了确定PAC分子的唾液结合区，利用聚合酶链式反应和表达载体pAX4a+构建了14个截短的PAC片段。用夹心法测定这些截短的PAC片段与人唾液成分的结合。在截短的PAC片段中，对应于PAC第39~864位残基和第39~1000位残基的片段显示出与唾液成分高度结合的能力。与第39~217残基、200~481残基、470~749残基、688~864残基相对应的较短重组片段不显示任何结合能力。与富含脯氨酸的重复区域(残基828至1000)相对应的片段直接与PAC蛋白结合。这些结果表明，PAC分子的39864位残基在表面蛋白与人唾液成分的结合中起着重要的作用，PAC蛋白富含脯氨酸的重复区域可能有助于PAC蛋白的自发聚集。
A 190-kDa surface protein antigen (PAc) of Streptococcus mutans binds to human salivary components. For detection of specific binding of the PAc protein to human salivary components, a simple sandwich assay was used. Microtiter plates precoated with recombinant PAc (rPAc), PAc fragments, or S. mutans whole cells were allowed to react with human whole saliva and then were incubated with biotinylated rPAc. The biotinylated rPAc bound to salivary components was detected by use of alkaline phosphatase-conjugated streptavidin and p-nitrophenylphosphate. In this assay, the binding of whole cells of S. mutans and purified rPAc to salivary components was confirmed. For determination of a saliva-binding region of the PAc molecule, 14 truncated PAc fragments were constructed by use of the polymerase chain reaction and an expression vector, pAX4a+. The binding of these truncated PAc fragments to human salivary components was determined by the sandwich assay. Among the truncated PAc fragments, fragments corresponding to residues 39 to 864 and residues 39 to 1000 of PAc showed a high ability to bind to salivary components. Shorter recombinant fragments corresponding to residues 39 to 217, residues 200 to 481, residues 470 to 749, and residues 688 to 864 did not exhibit any binding ability. The fragment that corresponds to a proline-rich repeating region (residues 828 to 1000) bound directly to the PAc protein. These results suggest that residues 39 864 of the PAc molecule are important in the binding of the surface protein to human salivary components, and the proline-rich repeating region of the PAc protein may contribute to spontaneous self-aggregation of the PAc protein.