变形链球菌AI-2活性测定及luxS基因同源重组质粒的构建

Detection of quorum-sensing pathway and construction of luxS gene allelic exchange plasmid of Streptococcus mutans

目的构建用于转化变形链球菌的luxS基因缺失的同源重组克隆载体。方法利用哈氏弧菌（Vibrio harveyi）BB170作为报告菌株，对变形链球菌在不同生长时期诱导生物发光的能力进行测定，然后分别PCR扩增变链菌luxS基因上下游片段及质粒PJT10的红霉素抗性基因片段，采用分子克隆技术将基因片段依次双酶切后连接人载体pUC19，构建luxS基因缺失的重组质粒，转化到大肠杆菌DH5α感受态细胞经筛选后，抽提重组质粒，进行酶切鉴定。结果变链菌国际标准株可诱导哈氏弧菌BB170的生物发光现象，提示变链菌中存在AI-2数量感应通路。构建的重组质粒pUCluxKO经PstI-BamHI双酶切，产物电泳在大约1000bp和5000bp处各见一条特异条带；经BamHI—KpnI双酶切，产物电泳在大约1500bp和4500bp处各见一条特异条带；经KpnI—EcoRI双酶切后，产物电泳在大约1000bp和5000bp处各见一条特异条带。结论变链菌luxS基因同源重组质粒构建成功，为进一步通过同源重组法构建变链菌luxS基因缺陷株奠定基础。

Objective To detect the AI-2 quorum-sensing pathway and construct the luxS g-ene allelic exchange plasmid of Streptococcus rnutans. Methods To dectect AI-2 pathway in Streptococcus rnutans, the Vibrio harveyi BB170 was used as reporter strain. The PCR fragments of the upstream and downstream regions of luxS and the Erythromycin resistance gene were amplified with the primers respetively, and these fragments were ligated into pUC19 vector with double endonuclease reaction sequentially, the ligated DNAs were transformed into Escherichia coli DH5α, then the reconstructed plasmids were isolated and identified by restricted endonuclease digestions. Results Streptococcus rnutans Ingbritt C could induce luminescene of BB170, suggesting the presense of AI-2 quorum sensing pathway in Streptococcus rnutans, and such stimulatory activity was maximal at the mid-log growth phase. The recombinant plasmid pUCluxKO was digested by PstI-BarnHI, and the digest product were 1000 bp and 5000 bp. When the pUCluxKO was digested by BamHI-Kpnl, the digest product were 1500 bp and 4500 bp. While it was digested by KpnI- EcoRI, the digest product were 1000 bp and 5000 bp. All PCR product was in a single belt respectively. Conclusions The recombinant plasmid was cloned effectively and can be used in the construction of S. mutans luxS mutant.