Huang Zheng-Wei, Liu Zheng
Dept. of Oral Medicine, The Ninth Affiliated Hospital of Shanghai Jiaotong University School of Medicine, Shanghai 200011, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2006 Feb;24(1):70-2.
It is reported that Streptococcus mutans luxS gene may have an important role in the interspecies quorum sensing system. To construct the S. mutans luxS gene knockout mutant, this research aim to construct the luxS gene allelic exchange plasmid.
The upstream and downstream flank DNA fragments of S. mutans luxS gene (Xup, Xdn)and the E. coli kanamycin resistance gene (Kana) were enriched by pfu DNA polymerase with "nest PCR" methods. These fragments were ligated into pBluescript SK (+) Phagemids vector with double endonuclease reaction sequentially.
With endonuclease reaction and DNA sequencing, it was proved that the objective plasmid, Xukd-pbsk, was constructed correctively and the kanamycin resistance gene could be expressed in vitro.
The S. mutans luxS gene allelic exchange plasmid is constructed correctively in this research and can be used in the future research of S. mutans luxS gene knockout mutant.
据报道变形链球菌luxS基因可能在种间群体感应系统中起重要作用。为构建变形链球菌luxS基因敲除突变体，本研究旨在构建luxS基因等位基因交换质粒。
采用“巢式PCR”方法，用pfu DNA聚合酶富集变形链球菌luxS基因的上下游侧翼DNA片段（Xup、Xdn）和大肠杆菌卡那霉素抗性基因（Kana）。这些片段依次通过双酶切反应连接到pBluescript SK(+)噬菌粒载体中。
通过酶切反应和DNA测序，证明目标质粒Xukd-pbsk构建正确，卡那霉素抗性基因可在体外表达。
本研究成功构建了变形链球菌luxS基因等位基因交换质粒，可用于后续变形链球菌luxS基因敲除突变体的研究。
