抑制消减杂交技术分析牙龈卟啉单胞菌基因差异初探

Studying the genetic differences of Porphyromonas gingivalis by suppression subtractive hybridization

目的:对比牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)低毒力株标准参考菌ATCC 33277与高毒力株W83之间的差异核苷酸序列,查找高毒力株W83缺失基因。方法:抑制消减杂交技术对比牙龈卟啉单胞菌低毒力株标准参考菌ATCC 33277与高毒力株W83的基因差异。以低毒力株ATCC 33277为tester,高毒力株W83株为driver,将提取的基因组DNA用内切酶RsaⅠ酶切,连接特殊设计的adaptor进行两次消减杂交和PCR扩增,并与TA克隆载体连接,转化到JM109中,建立差异消减文库,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62个片段大小为125～632bp的阳性克隆基因片段。结论:这些差异基因为研究牙周病的发生和发展提供重要线索。

Objective：To identify the differential genes in Porphyromonas gingivalis minimally toxic strain ATCC 33277 and highly toxic strain W83.Methods：Porphyromonas gingivalis minimally toxic strain ATCC 33277（tester） and highly toxic strain W83（driver） were compared with each other by using suppression subtractive hybridization（SSH）.The chromosomal DNAs were purified from P gingivalis ATCC 33277 and P.gingivalis W83,and digested by restriction enzyme RsaⅠ.The tester DNA samples were separated and ligated with adaptor 1 and adaptor 2R.The ligation efficiency was performed to verify that at least 25% of tester DNA fragments had adaptors on both ends.Two subtractive hybridization and PCR profile were performed.Tester-specific DNAs were also selectively amplified.Then the subtraction efficiency analysis was performed.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：The products were obtained through subtractive hybridization and PCR amplification twice.The experimental PCR subtraction products with some distinct bands were different from unsubtraction products.The distinct bands were rich in Porphyromonas gingivalis ATCC 33277,while absent from P.gingivalis highly toxic strain W83.Subtractive library which had high subtractive efficiency was successfully set up and 119 positive clones were screened by SSH.The fragments from 125 bp to 632 bp were rich in Porphyromonas gingivalis minimally toxic strain ATCC 33277,while absent from P.gingivalis W83.Conclusion：These genes may provide an important clue for studying the mechanism of occurrence and development of periodontal disease.