›› 2019, Vol. 39 ›› Issue (4): 306-310.

• 基础研究 • 上一篇    下一篇

牙龈卟啉单胞菌clpP基因缺失株和回补株的构建和鉴定

何露1,王宏媛1,张茹2,侯本祥2,李红2   

  1. 1. 首都医科大学附属北京口腔医院
    2. 首都医科大学附属北京口腔医院牙体牙髓科
  • 收稿日期:2018-10-23 修回日期:2018-11-25 出版日期:2019-04-28 发布日期:2020-01-13
  • 通讯作者: 李红 E-mail:lihongdentist@163.com
  • 基金资助:
    ClpP蛋白酶对牙龈卟啉单胞菌生物膜调控机制的研究

Construction and identification of the clpP deletion strains and complemented strains in Porphyromonas gingivalis

  • Received:2018-10-23 Revised:2018-11-25 Online:2019-04-28 Published:2020-01-13
  • Contact: Li 无Hong E-mail:lihongdentist@163.com

摘要: 目的 构建并鉴定牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)W83的丝氨酸蛋白酶(caseinolytic protease,Clp)基因缺失株和回补株,为探索clpP基因在P.g致病过程中的作用和机制奠定基础。方法 设计clpP基因的引物对,PCR扩增其上下游同源片段,克隆入质粒pUC18中,插入红霉素抗性基因ermB作为筛选标记,构建重组质粒,电转化入P.g W83构建缺失株;PCR扩增clpP基因片段与重组质粒pUC18-ΔclpP-ermB连接,电转化入P.g W83缺失株中构建回补株。采用PCR和酶切电泳鉴定序列的正确性,利用抗性培养基筛选高表达菌株。结果 clpP基因克隆成功并顺利导入质粒pUC18中,P.g W83的clpP基因缺失株和回补株构建成功,并能在体外稳定传代。结论 本研究运用同源重组技术构建了P.g W83的clpP基因缺失株,并建立了以pUC18质粒为载体的clpP基因回补方法,为进一步研究clpP基因在P.g致病过程中的作用打下了基础。

关键词: clpP基因, 牙龈卟啉单胞菌, 缺失重组株, 回补重组株

Abstract: Abstract: Objective The caseinolytic protease (clp) gene deletion strains and complemented strains of Porphyromonas gingivalis (P.g) W83 were constructed and identified in order to make a foundation for exploring the role and mechanism of clpP gene in the pathogenesis of P.g W83. Methods The primer pair of clpP gene were designed and its upstream and downstream homologous fragments were amplified by PCR, which were cloned into plasmid pUC18. And the erythromycin resistance gene ermB from plasmid pMG36e was inserted as a screening marker to construct a recombinant plasmid,which was electrotransfered into P.g W83 to get the corresponding clpP deficient recombinant strain. The clpP gene fragment was amplified by PCR and ligated with the recombinant plasmid pUC18-△clpP-ermB and electrotransfered into the recombinant strain of P.g so as to construct the corresponding complemented recombinant strain. PCR and electrophoresis were used to identify the correctness of the sequence, and the high-expression strain was screened and selected using a resistant medium. Results The cloned clpP gene fragment was successfully cloned and introduced into the plasmid vector pUC18. The clpP gene deletion strain and the complemented strain of P.g W83 were successfully constructed and could be stably passaged in vitro. Conclusion In this study, the clpP gene deletion strain of P.g W83 was constructed using homologous recombination technology, and the method of using pUC18 plasmid as a vector to construct the complemented strains was established. What we did would help to study the role of clpP gene in the pathogenesis of P.g in future.

Key words: clpP, porphyromonas gingivalis, deletion strain, complemented strain

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