Basic studies on development of DNA diagnosis for periodontal disease associated bacteria

我们进行了牙龈卟啉单胞菌核糖体RNA的克隆。该克隆DNA与几种牙周病相关细菌的DNA杂交。由于牙龈假单胞菌的rDNA与其他牙周病相关细菌的rDNA具有同源性，因此很难确定基因的特异性区域。因此，我们尝试分离到牙龈假单胞菌200 kDa特异性蛋白与大肠杆菌麦芽糖结合蛋白主抗原区融合蛋白的基因克隆。重组克隆产生65 kDa的融合蛋白。融合蛋白可经直链淀粉区域蛋白亲和柱层析纯化。用凝血因子Xa将融合蛋白洗脱到65 kDa蛋白和麦芽糖结合蛋白的主要抗原区后，65 kDa融合蛋白可以在同一亲和层析柱上洗脱和回收。这些结果表明，成功地将牙周炎螺旋体200 kDa特异性蛋白的主要抗原区克隆到pMal载体中，该基因克隆可用于大规模生产用于牙周炎血清学诊断的抗原。我们还尝试了罗氏普氏杆菌、牙髓螺旋体、齿密螺旋体、直弯杆菌等牙周病相关细菌的特异性抗原的基因克隆。由于成人牙周炎患者的血清可识别每一种重组蛋白，这些蛋白将成为牙周病免疫学诊断的有用抗原。在另一种方法中，我们尝试制作DNA探针用于牙周病的DNA诊断。我们分离到了仅与牙龈假单胞菌染色体DNA杂交的DNA探针。DNA探针用地高辛标记，而不是一直用于标记DNA的同位素标记。非同位素标记的DNA探针可作为牙周疾病常规诊断的有用工具。

We have done cDNA cloning of ribosomal RNA of Porphylomonas gingivalis. The clone DNA hybridized with DNA from several periodontal disease associated bacteria. As rDNA of P. gingivalis had homology with other periodontal disease associated bacteria, it is difficult to identify the gene specific regions. We therefore attempted to isolate a gene clone encoding the fusion protein of the major antigenic region of 200 kDa specific protein of P. gingivalis and E. coli maltose binding protein. A recombinant clone produced 65 kDa fusion protein. The fusion protein could be purified amylose regin affinity column chromatography. After clevage of the fusion protein to the major antigenic region of 65 kDa protein and the maltose binding protein using Factor Xa, 65 kDa fusion protein could be eluted and be recovered by the same affinity column. These data suggest that the major antigenic region of P. gingivalis 200 kDa specific protein was successfully cloned in pMal vector plasmid and the gene clone is useful for large quantity production of antigen for serodiagnosis of P. gingivalis infection.We also have attempted gene cloning of specific antigens of other periodontal disease associated bacteria such as Prevotella loescheii, P. endodontalis, Treponema denticola, Camphylobacter rectus. Since human sera from adult periodontitis patients recognized each recombinant protein, these proteins will be useful antigens for the development of immunological diagnosis of periodontal diseases.In another approach we have attempted to make DNA probe to use the DNA diagnosis of periodontal diseases. We have isolated DNA probe hybridized with only P. gingivalis chromosomal DNA. The DNA probe was labeled with digoxigenin instead of labeling with isotope which has been using for labeling DNA. The non-isotope labeled DNA probe must be useful tool for routinely use in diagnosis of periodontal diseases.