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

对牙龈卟啉单胞菌核糖体RNA进行了cDNA克隆。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具有同源性，因此很难确定其基因特异性区域。因此，我们试图分离一个编码牙龈假单胞菌200 kDa特异性蛋白主抗原区融合蛋白和大肠杆菌麦芽糖结合蛋白的基因克隆。重组克隆产生65kda的融合蛋白。融合蛋白可通过直链淀粉区亲和柱层析纯化。用Xa因子将融合蛋白切割到65 kDa蛋白和麦芽糖结合蛋白的主抗原区后，65 kDa融合蛋白可以被洗脱并通过同一亲和柱回收。这些数据表明，在pMal载体质粒上成功克隆了牙龈假单胞菌200 kDa特异性蛋白的主要抗原区，该基因克隆为大批量生产牙龈假单胞菌感染血清诊断抗原提供了依据。我们还尝试了其他牙周病相关细菌的特异性抗原的基因克隆，如罗氏普雷沃氏菌、牙髓假单胞菌、牙密螺旋体、直弯曲杆菌。由于成人牙周炎患者的血清能够识别每种重组蛋白，因此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，我们尝试用DNA探针来诊断牙周病。我们分离的DNA探针只与牙龈假单胞菌染色体DNA杂交。DNA探针用地高辛标记，而不是用同位素标记DNA。非同位素标记DNA探针必须是常规诊断牙周病的有用工具。

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.