中华皮肤科杂志 ›› 2003, Vol. 36 ›› Issue (5): 264-266.

• 论著 • 上一篇    下一篇

耐药淋球菌差异DNA消减文库的构建及初步筛选

季明春, 严华, 陈红菊, 申厚凤   

  1. 扬州大学医学院, 微生物学及免疫学教研室, 225001
  • 收稿日期:2002-10-23 出版日期:2003-05-15 发布日期:2003-05-15
  • 通讯作者: 季明春(E-mail:mcji@yzu.edu.cn) E-mail:mcji@yzu.edu.cn

Cloning and Identifying the Differential Genes in Resistant Neisseria Gonorrhoeae

JI Ming-chun, YAN Hua, CHEN Hong-ju, SHEN Hou-feng   

  1. Department of Microbiology and Immunology, Yangzhou University, Yangzhou 225001, China
  • Received:2002-10-23 Online:2003-05-15 Published:2003-05-15

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摘要： 目的 克隆和研究耐药性淋球菌相关基因。方法 从耐药淋球菌株RSM292C4和淋球菌标准参考菌株WHO-A细胞中提取DNA,经RsaⅠ酶切后,应用抑制性消减杂交技术构建耐药性淋球菌差异DNA消减文库并进行初步筛选。结果 耐药淋球菌差异DNA消减文库经扩增获得约2500个白色克隆,随机挑取96个白色克隆,进行PCR扩增鉴定,发现克隆中插有100~600bp大小的片段。分别以RsaI酶切样本和参照DNA为探针,与随机挑取的96个白色菌落质粒作点杂交,有5个克隆仅能与样本DNA探针杂交,而不能与参照DNA探针杂交,表明它们为RSM292C4基因组DNA所特有,可能代表某些淋球菌新基因。对这5个克隆插入的DNA片段进行测序,经送基因库和淋球菌基因组部分测序基因库检索分析,发现5个片段均为未知新序列。结论 淋球菌耐药菌株DNA消减文库的构建为进一步筛选、克隆淋球菌耐药的新基因奠定了基础。

关键词: 奈瑟球菌,淋病, 药物耐受,细菌, 抑制性消减杂交, 基因文库, 序列分析,DNA

Abstract: Objective To clone and study the antimicrobial-resistant gene in Neisseria gonorrhoeae.Methods The gene library,which contains the differential genes of antimicrobial resistant strains and standard reference strains of Neisseria gonorrhoeae,was constructed using a technique known as suppression subtractive hybridization(SSH).Then the antimicrobial resistance associated genes were cloned and analyzed.Results Subtractive gene library in antimicrobial-resistant Neisseria gonorrhoeae was successfully constructed,which contains 2500 positive clones.Sequence analysis was performed on5clones.The sequences of these five clones were unknown previously.Conclusions The subtractive DNA library is successively constructed which may provide an important clue for studying the mechanism of antimicrobial resistance in Neisseria gonorrhoeae.

Key words: Neisseria gonorrhoeae, Drug resistance,bacterial, Suppression subtractive hybridiza-tion, Sequence analysis,DNA, Gene library