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目的比较牙龈卟啉单胞菌(Porphyromonasgingivalis,P·gingivalis)高毒力株W83与低毒力株标准参考菌ATCC33277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ATCC33277的基因差异。以高毒力株W83为被检菌,低毒力株ATCC33277为参考菌,将提取的基因组DNA用内切酶RsaⅠ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ATCC33277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。
OBJECTIVE: To compare the differential genes between the Porphyromonas gingivalis (P · gingivalis) highly virulent strain W83 and the low virulent strain reference reference strain ATCC33277. Methods The genetic differences between Pseudomonas gingivalis virulent strain W83 and low virulent strain reference strain ATCC33277 were compared by inhibition subtractive hybridization (SHH). The highly virulent strain W83 was used as the tested bacteria and the low virulent strain ATCC33277 as the reference bacteria. The extracted genomic DNA was digested with restriction endonuclease Rsa Ⅰ, and then the specially designed linkers were subjected to two subtractive hybridizations and PCR amplification to obtain the subtracted The mixture was ligated with the TA cloning vector, transformed into JM109, and a subtractive library was established. Positive clones were identified by PCR screening, and then some of the fragments were sequenced and homologous analyzed. Results A total of 36 positive clones with size of 88 ~ 372bp were obtained by SSH screening. Conclusion The molecular genetic differences between Pseudomonas gingivalis virulent strain W83 and the standard strain ATCC33277 were studied from the perspective of whole genome. It was a screening target gene of Porphyromonas gingivalis and the target of prevention, diagnosis and treatment of periodontal disease Provide evidence.