牙龈卟啉单胞菌不同毒力株基因差异的比较研究

目的比较牙龈卟啉单胞菌（Porphyromonas gingivalis，Pgingivalis）高毒力株W83与低毒力株标准参考菌ATCC 33277之间的差异基因。方法采用抑制消减杂交技术（SHH）对比牙龈卟啉单胞菌高毒力株W83与低毒力株标准参考菌ATCC 33277的基因差异。以高毒力株W83为被检菌，低毒力株ATCC 33277为参考菌，将提取的基因组DNA用内切酶Rsa Ⅰ酶切，连接特殊设计的接头进行两次消减杂交和PCR扩增，得到消减混合物，与TA克隆载体连接，转化到JM109中，建立消减文库，经PCR筛选鉴定阳性克隆，进而对部分片段进行测序和同源分析。结果经SSH筛选鉴定得到36个片段大小为88～372bp的阳性克隆基因片段。结论从全基因角度研究牙龈卟啉单胞菌高毒力株W83与标准株ATCC 33277之间的分子遗传差异，为牙龈卟啉单胞菌致病基因的筛选及今后牙周病预防、诊治的靶标提供依据。

Comparison between genes of highly toxic strain and minimally toxic strain of Porphyromonas gingivalis

OBJECTIVE: To identify the differential genes in Porphyromonas gingivalis (P.gingivalis) highly toxic strain W83 and minimally toxic strain ATCC 33277. METHODS: Using suppression subtractive hybridization (SSH) to compare P.gingivalis highly toxic strain W83 (tester) and minimally toxic strain ATCC 33277 (driver). The chromosomal DNAs were purified from P.gingivalis W83 and P.gingivalis ATCC 33277, and digested by restriction enzyme RsaI. The tester DNA samples were separated and ligated with adaptor 1 and adaptor 2R. Two subtractive hybridization and PCR profile were performed. Tester-specific DNAs also were selectively amplified. The mixture of subtracted DNA fragments were ligated with pMD-18T vector and transformed to competent cells E.coli JM109. The differential subtraction library was established. The positive clones were identified by PCR and then sequenced, and searched homologically. RESULTS: Subtractive library which had high subtractive efficiency was successfully set up and 36 positive clones were screened by SSH. The fragments from 88 bp to 372 bp were enriched in P.gingivalis highly toxic strain W83 sequences which were absent from P.gingivalis ATCC 33277. Through dot blot analysis confirmed that all these fragments were present in P.gingivalis W83 but absent from ATCC 33277. The GenBank homology search indicated that among them, several genes were associated with two paralogous regions of the chromosome; Some genes are associated with evasion of P.gingivalis W83; Another gene was related to antibiotic resistance and the products of some genes were virulence and acquisition of peptides. CONCLUSIONS: Comparative whole-genome analysis of highly toxic and minimally toxic strains of P.gingivalis has identified the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277. These genes may provide an important clue for studying the mechanism of occurrence and development of periodontal disease.