Analysis of gene of Granulicatella adiacens fibronectin-binding protein and application of the gene for clinical diagnosis.

颗粒菌纤连蛋白结合蛋白基因分析及其临床诊断应用。

• 批准号: 12470464
• 负责人: INOUE Masakazu
• 金额: $ 7.68万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470464/
• 关键词: Nutritionally variant streptococci (NVS); Granulicatella adiacens; Oral indigenous bacteria; Infective endocarditis; Fibronectin; Gene cloning; 2', 3' -cyclic nucleotide 2' -phosphodiesterase; Polymerase chain reaction (PCR); 細胞外基質タンパク質; DNAライブラリ

A gene encoding a protein with an ability of fibronectin binding was cloned from Granulicatella adiacens ATCC 49175T. DNA sequence revealed that the gene was located from 806 to 2419 on the 2540-bp EcoRI-XbaI fragment from the genomic DNA of G adiacens. The deduced amino acid sequence showed 54% identity to Lactococcus lactis 2', 3' -cyclic nucleotide 2' -phosphodiesterase (EC 3.1.4.16). An antibody specific to the recombinant protein was prepared in mice, but it was not able to bind to the cell surface of G adiacens, suggesting that the protein is not exposed on the surface of the organism. A set of PCR primers was designed according to the DNA sequences of the gene and DNA from various species were subjected to amplification. A 0.52-kb PCR product was yielded only from strains of G adiacens and closely related species produce no product. Clinical specimens of dental plaques from healthy volunteers produced a single 0.52-kb product, as did the type strain G adiacens. These results suggest that the PCR primers are applicable to detection and identification of G adiacens for clinical diagnosis.

从 Granulicatella adiacens ATCC 49175T 中克隆了编码具有纤连蛋白结合能力的蛋白质的基因。 DNA序列显示该基因位于G adiacens基因组DNA的2540 bp EcoRI-XbaI片段上的806至2419。推导的氨基酸序列显示与乳酸乳球菌2',3'-环核苷酸2'-磷酸二酯酶(EC 3.1.4.16)有54％的同一性。在小鼠体内制备了针对重组蛋白的特异性抗体，但它无法与 G adiacens 的细胞表面结合，表明该蛋白并未暴露在生物体表面。根据该基因的DNA序列设计一套PCR引物，对不同物种的DNA进行扩增。仅从 G adiacens 菌株中产生 0.52 kb PCR 产物，而密切相关的物种不产生产物。来自健康志愿者的牙菌斑临床样本产生了单一 0.52 kb 的产物，G adiacens 型菌株也是如此。这些结果表明PCR引物适用于G adiacens的检测和鉴定以用于临床诊断。