牙龈卟啉单胞菌prtH基因分布与多态性研究

Deletion and point mutations exist in the haemagglutinins/adhesions domain of prtH gene of Porphyromonas gingivalis clinical isolates

目的 分析牙龈卟啉单胞菌 (P .gingivalis)prtH基因的遗传多态性 ,从而了解细菌变异与其对牙周致病作用的相关性。方法 以PCR技术从牙周炎患者口腔中分离的P .gingivalis进行prtH基因扩增 ;采用斑点杂交法 ,以生物素标记prtH基因为探针 ,与以引物B进行PCR扩增为阳性的P .gingivalis基因组DNA杂交 ;对PCR产物进行AluⅠ限制性酶切和DNA测序分析。结果 以prtH基因A、B引物对 14 0株临床分离株进行PCR扩增 ,分别在 76例 (引物A)和 117例 (引物B)出现阳性扩增产物 ,阳性率分别为 5 4 .2 %、84 %。对 34例引物B扩增阳性的P .gingivalisDNA进行点杂交检测 ,阳性吻合率为 82 .4 %。与GenBank中菌株ATCC332 77(U15 2 82 )和W83(L2 74 83)的prtH基因序列相比 ,5株P .gingivalisPCR扩增产物的测序存在着缺失和点突变。结论 在慢性牙周炎患者临床P .gingivalis分离菌株中存在着prtH基因的遗传多态性 。

Objective Porphyromonas gingivalis ( P. gingivalis ) is thought to be a major etiologic bacterium associated with the initiation and progression of human periodontitis. The aim of the present study was to investigate the genetic diversity of P. gingivalis prt H gene. Methods Polymerase chain reaction(PCR) was applied to P. gingivalis strains, including reference strain P. gingivalis ATCC33277 and 140 clinical strains isolated from 24 unrelated Chinese periodontitis patients. To illustrate the prt H heterogeneity among P. gingivalis strains, the genomic DNAs were extracted and screened by PCR with two pairs of specific primers, and the PCR products were digested with restriction endonucleases AluⅠ. Using the biotin-labeled prt H sequence as probe, Dot blot hybridization was used to confirm the existence of prt H. To evaluate the sequence heterogeneity of the P. gingivalis prt H genes, sequence analysis of five prt H protease gene regions of clinical isolates was performed. Results Using PCR with two pairs of specific primers, 54.2% (primer A) and 84% (primer B) showed the positive amplified fragment among 140 strains. Dot blot hybridization revealed that 82.4% PCR products were positive. Restriction endonucleases analyses showed that all the PCR products were P. gingivalis prt H patterns. Sequence analysis verified that there were deletion and point mutations in five PCR products, compared with the reference strain P. gingivalis ATCC33277 and W83. Conclusion The results demonstrated genetic diversity existed among these clinical strains isolated from Chinese periodontitis patients.