牙龈卟啉单胞菌prtH基因克隆及其多态性的研究

目的：建立牙龈卟啉单胞菌（Porphyromonas gingivalis,P.g)prtH全基因序列的克隆株，分析5株不同P.g菌株中prtH基因多态性。方法：利用PCR技术获得prtH全基因序列，克隆至载体pGEM-T;设计3对引物，用PCR的方法扩增prtH基因的不同区域；以生物素标记prtH基因序列为特异性探针，用斑点杂交和Southern印迹杂交进一步分析prtH基因的多态性。结果：酶切分析和DNA测序证实重组质粒pGEM-T-prtH的插入片段为prtH基因序列；核酸分子杂交显示菌株W 381和ATCC 33277杂交类型一致，菌株ATCC 49417和菌株14－3－2呈现各自不同的杂交条带，而菌株47A－1中未检测到prtH基因。结论：不同P.g菌株中prtH基因序列存在差别，这种基因多态性提示不同P.g菌株毒力大小可能存在差异。所建立的PCR扩增技术可以敏感地检测prtH基因的存在。

Cloning and polymorphism analysis of prtH gene from Porphyromonas gingivalis

Objective To clone the prtH gene from Porphyromonas gingivalis (P g) ATCC 33277 and analyze the polymorphism of prtH gene from 5 strains of P g in order to explore the relationship between P g and periodontitis Methods Using PCR, the prtH was amplified and cloned into pGEM T vector To illustrate the prtH polymorphism among P g strains, the genomic DNAs were extracted and screened by PCR with three pairs of specific primers, dot blot and Southern blot hybridization using the biotin labeled prtH sequence as probe Results Recombinant DNA pGEM T prtH was verified by restriction endonuclease and sequence assay Strain W 381 and ATCC 33277 showed the identical results in PCR and hybridization assays, whereas strain ATCC 49417 and 14 3 2 revealed individual hybridization patterns Strain 47A 1 seemed even not to contain prtH gene Conclusions Different prtH gene sequences exist in different P g strains This polymorphism may indicate various potential virulent effects during the infection and pathogenesis Established PCR protocol is sensitive for identification of prtH gene