口腔医学研究

口腔医学研究 ›› 2018, Vol. 34 ›› Issue (5): 490-494.DOI: 10.13701/j.cnki.kqyxyj.2018.05.008

• 口腔微生物学研究 • 上一篇    下一篇

变形链球菌耐氟菌株gtfB基因失活菌株的构建

李文月, 高爽, 张志鹰, 张红, 超博, 武玉凤, 李贺, 王春萌, 张志民*   

  1. 吉林大学口腔医院牙体牙髓科 吉林 长春 130021
  • 收稿日期:2017-09-29 出版日期:2018-05-28 发布日期:2018-05-29
  • 通讯作者: 张志民,E-mail:zhangzm1964@sina.com
  • 作者简介:李文月(1991~ ),女,河北唐山人,硕士在读,主要研究龋病黏附机制。

Construction of Streptococcus Mutans Fluoride-resistant Strain with GtfB Gene Inactive.

LI Wen-yue, GAO Shuang, ZHANG Zhi-ying, ZHANG Hong, CHAO Bo, WU Yu-feng, LI He, WANG Chun-meng, ZHANG Zhi-min*   

  1. Department of Endoclontics School of Stomatology, Jilin University, Changchun 130021, China
  • Received:2017-09-29 Online:2018-05-28 Published:2018-05-29

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摘要： 目的:构建变形链球菌(S.mutans)耐氟菌株UA159-FR中gtfB基因失活株,为研究耐氟菌株gtfB基因的功能奠定基础。方法:培养UA159-FR并以其为模板扩增gtfB基因上游、下游同源臂片段,以质粒pEGFP-N1为模板扩增kan基因;通过重叠延伸聚合酶链反应法(overlap extension polymerase chain reaction,OE-PCR)获取上述3个片段的同源重组片段;与pEASY-Blunt Cloning Vector连接形成重组质粒后,进行PCR鉴定和测序鉴定;将重组片段电转化入UA159-FR感受态细胞中得到gtfB基因失活菌株,并进行PCR鉴定。结果:经PCR鉴定和测序鉴定,含有gtfB基因重组片段的重组质粒构建成功;经PCR鉴定,UA159-FR的gtfB基因失活株构建成功。结论:成功构建了含有gtfB基因重组片段的重组质粒和S.mutans耐氟菌株UA159-FR的gtfB基因失活株,可用于gtfB基因功能的研究。

关键词: 变形链球菌耐氟菌株, gtfB基因, 重叠延伸聚合酶链反应, 同源重组, 基因失活

Abstract: Objective: To construct Streptococcus mutans (S.mutans) fluoride-resistant strain UA159-FR with gtfB gene inactive to investigate the function of gtfB gene in fluoride-resistant strain. Methods: The S.mutans fluoride-resistant strain UA159-FR was cultured and used as a template to amplify the upstream and downstream homologous arm fragments of the gtfB gene. The kan gene was amplified by using the plasmid pEGFP-N1 as a template. Homologous recombination fragments of three fragments were obtained by overlap extension polymerase chain reaction (OE-PCR) and ligated with pEASY-Blunt Cloning Vector to form recombinant plasmids. The recombinant plasmids were identified by PCR and sequencing. The recombinant fragment was electrotransformed into UA159-FR competent cells to obtain the inactivated strain and identified by PCR. Results: The recombinant plasmids were successfully constructed via being identified by PCR and sequencing. It was identified by PCR that UA159-FR with gtfB gene inactive was obtained. Conclusion: The recombinant fragments of gtfB gene of S.mutans with its recombinant plasmids and the S.mutans fluoride-resistant strain UA159-FR with gtfB gene inactive were successfully constructed and could be used to study the function of gtfB gene.

Key words: Fluoride-resistant streptococcus mutans, GtfB gene, Overlap extension polymerase chain reaction, Homologous recombination, Gene inactivation