Development of simple and high sensitive method for caries risk diagnosis by molecular genetics

建立简单、高灵敏度的分子遗传学龋齿风险诊断方法

• 批准号: 14571954
• 负责人: SHIBATA Yukie
• 金额: $ 2.18万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571954/
• 关键词: caries; Streptococcus mutans; serotype-specific antigen; serotype-specific glucose side-chain formation; PCR; prevalence of serotypes; caries experience; グルコース側鎖形成

First, we cloned the genes responsible for the serotype c-specific glucose side-chain formation of rhamnose-glucose polysaccharide (RGP) in Streptococcus mutans. There were five ORFs in this region, and two novel genes (rgpH and rgp) were required for glucose side-chain formation. RGP produced in E.coli containing genes required for rhamnan synthesis and rgpH reacted with serotype c-specific rabbit antiserum, whereas rgpl did not produce glucose side chains. The addition of rgpl was drastically decreased the reactivity of RGP with rhamnan-specific antiserum. These results suggest that rgpH may be a unique glucosyltransferase for glucose side chain formation of RGP, whereas rgpl may be a regulatory gene which involve in the up-regulation of glucosylation. Next, the corresponding chromosomal regions were isolated from serotype e and f strains and characterized. The rgpA-F homologs were well conserved among the three serotypes. By contrast, the regions downstream from the rgpF homolog differed considerably among the three serotypes. Replacement of these regions in the different serotype strains converted their serotypic phenotypes, suggesting that these regions participated in serotype-specific glucose side-chain formation in each serotype strain. Based on the differences among the DNA sequences of these regions, a PCR method was developed to determine serotypes.S.mutans was isolated from 198 of 432 preschool children (3〜4 years old). The serotypes of S.mutans in all but one were identified with serotyping PCR. Serotype c predominated (84.8%), serotype e was the next most common (13.3%), and serotype f occurred rarely (1.9%) in Japanese preschool children. Caries experience in the group with a mixed infection of multiple serotypes of S.mutans was significantly higher than that in the group with a mono infection of a single serotype.

首先，我们克隆了变形链球菌中鼠李糖-葡萄糖多糖(RGP)的C型特异性葡萄糖侧链形成相关基因。该区域有5个ORF，葡萄糖侧链的形成需要两个新基因(rgpH和rgP)。在含有鼠李糖合成所需基因的大肠杆菌中产生的RgP和rgpH能与c型特异性兔抗血清反应，而rgp1不产生葡萄糖侧链。Rgp1的加入显著降低了RGP与鼠李糖特异性抗血清的反应性。这些结果表明，rgpH可能是RGP葡萄糖侧链形成所特有的糖基转移酶，而rgp1可能是参与糖基化上调的调控基因。接下来，从e型和f型菌株中分离相应的染色体区域，并对其进行鉴定。RgpA-F同源基因在三个血清型之间保存良好。相反，rgpF同源基因下游的区域在三种血清型之间有很大差异。不同血清型毒株中这些区域的替换转换了它们的血清型表型，表明这些区域参与了每个血清型毒株的血清型特异性葡萄糖侧链的形成。从432名3~4岁学龄前儿童中分离到198株变形链球菌，根据DNA序列的差异，建立了一种检测变形链球菌血清型的方法。用血清分型聚合酶链式反应对除1株外的所有菌株进行了血清型鉴定。日本学龄前儿童以c型为主(84.8%)，其次为e型(13.3%)，f型很少(1.9%)。变形链球菌混合感染组患龋率明显高于单一血清型感染组。

项目成果

Y.Shibata et al.: "Analysis of loci required for determination of serotype-specific antigenicity in Strepococcus mnutans and its clinical utilization"Journal of Clinical Microbiology. 41-9. 4107-4112 (2003)

Y.Shibata 等人：“分析变形链球菌血清型特异性抗原性所需的基因座及其临床应用”临床微生物学杂志。

K.Ozaki et al.: "A novel mechanism for glucose side-chain formation in rhamnose-glucose polysaccharide synthesis"FEBS Letters. 532. 159-163 (2002)

K.Ozaki 等人：“鼠李糖-葡萄糖多糖合成中葡萄糖侧链形成的新机制”FEBS Letters。

Kazuhisa Ozaki et al.: "A novel mechanism for glucose side-chain formation in rhamnose-glucose polysaccharide synthesis"FEBS Letters. 532. 159-163 (2002)

Kazuhisa Ozaki 等人：“鼠李糖-葡萄糖多糖合成中葡萄糖侧链形成的新机制”FEBS Letters。

Yukie Shibata et al.: "Analysis of loci required for determination of serotype-specific antigenicity in Streptococcus mutans and its clinical utilization"Journal of Clinical Microbiology. 41・9. 4107-4112 (2003)

Yukie Shibata等：“变形链球菌血清型特异性抗原性测定所需基因座的分析及其临床应用”临床微生物学杂志41・9 4107-4112（2003）。