敲除牙龈卟啉单胞菌菌毛次要组分FimCDE的载体构建

上海口腔医学 ›› 2013, Vol. 22 ›› Issue (6): 623-627.

敲除牙龈卟啉单胞菌菌毛次要组分FimCDE的载体构建

黎景景^1,2，高鑫²，李宽钰²，王文梅¹

(1.南京大学医学院附属口腔医院口腔黏膜病科，江苏南京 210008；2.南京大学医学院江苏省医学分子技术重点实验室，江苏南京 210093)

收稿日期:2013-01-14 修回日期:2013-05-13 出版日期:2013-04-12 发布日期:2013-04-12
通讯作者: 王文梅，Tel：025-83620103，Fax：025-83620202,E-mail:wangwenmei0102@163.com
作者简介:黎景景 (1990-)，男，硕士研究生，医师，E-mail:lijingjing900622@163.com
基金资助:
国家自然科学基金(81070839)；江苏省医学领军人才与创新团队项目(LJ201110)；南京市科技发展计划项目(YKK06115)；南京市医学科技发展重点项目(ZKX1030

Construction of recombinant for generation of Porphyromonas gingivalis minor accessory proteins FimCDE deficient strain

LI Jing-jing ^1,2, GAO Xin ², LI Kuan-yu ², WANG Wen-mei ¹

1.Department of Oral Medicine, School of Stomatology, Nanjing University & Nanjing Stomatological Hospital. Nanjing 210008; 2.Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University. Nanjing 210093, Jiangsu Province, China

Received:2013-01-14 Revised:2013-05-13 Online:2013-04-12 Published:2013-04-12
Supported by:
Supported by National Natural Science Foundation of China (81070839). Team Project of Medical Leaders in Talent and Innovation of Jiangsu Province (LJ201110), Science and Technology Development Plan of Nanjing City(YKK06115) and Medical Science and Technology Development Project of Nanjing City(ZKX1030).

摘要/Abstract

摘要： 目的：构建重组质粒pPHU281-C-Spec-E，用于敲除菌毛蛋白次要组分基因fimCDE。构建fimCDE缺陷的牙龈卟啉单胞菌，为进一步研究FimCDE在感染中的作用奠定基础。方法：厌氧罐培养牙龈卟啉单胞菌菌株 ATCC 33277，提取基因组DNA后，PCR扩增获得含有人工设计的酶切位点的fimC基因的上游片段及fimE的下游片段，将其克隆到自杀质粒pPHU281内，命名为pPHU281-C-E；再将抗大观霉素的抗性基因插入到C及E片段之间，最终获得重组质粒pPHU281-C-Spec-E。结果：酶切及DNA序列分析验证重组质粒pPHU281-C-Spec-E构建成功。结论：成功构建了含有牙龈卟啉单胞菌菌毛蛋白次要组分基因fimC上游及fimE下游片段的重组质粒pPHU281-C-Spec-E,可用于构建菌毛蛋白次要组分FimCDE缺陷的的突变体。

关键词: 牙龈卟啉单胞菌, 菌毛蛋白次要组分FimCDE突变体, 载体构建

Abstract: PURPOSE: To construct a recombinant plasmid containing the upstream of fimC and downstream of fimE of Porphyromonas gingivalis, designated as pPHU281-C-Spec-E, which may be further used to knock out fimCDE gene to determine the role of FimCDE in the infection by P. gingivalis. METHODS: DNA fragments were generated by PCR with the genomic DNA of P. gingivalis strain ATCC 33277 as the template. The upstream fragment of fimC (fragment C) and downstream fragment of fimE (fragment E) were cloned into the suicide plasmid pPHU281 to generate plasmid pPHU281-C-E. The spectinomycin resistance gene was inserted between fragment C and E to construct plasmid Pphu281-C-Spec-E. The recombinant plasmid was verified by restriction enzyme digestion and DNA sequencing. RESULTS: The recombinant plasmid pPHU281-C-Spec-E was successfully constructed, which was ready for generation of FimCDE-knockout mutant of P. gingivalis. CONCLUSIONS: The recombinant plasmid pPHU281-C-Spec-E is a tool for construction of FimCDE deficient mutant of P. gingivalis.

Key words: Porphyromonas gingivalis, FimCDE mutant, Recombinant plasmid

中图分类号:

Q782

黎景景, , 高鑫 , 李宽钰 , 王文梅. 敲除牙龈卟啉单胞菌菌毛次要组分FimCDE的载体构建[J]. 上海口腔医学, 2013, 22(6): 623-627.

LI Jing-jing , GAO Xin , LI Kuan-yu , WANG Wen-mei. Construction of recombinant for generation of Porphyromonas gingivalis minor accessory proteins FimCDE deficient strain[J]. Shanghai Journal of Stomatology, 2013, 22(6): 623-627.

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