LuxS基因缺失的变形链球菌突变株的构建及鉴定  

Construction and identification of a LuxS-deleted mutant strain of S. mutans

作　　者：于丹妮[1] 韩福胜[1] 韩玉植[1] 陈杰[1] 

机构地区：[1]天津医科大学第二医院口腔科,300211

出　　处：《中华微生物学和免疫学杂志》2008年第3期198-202,共5页Chinese Journal of Microbiology and Immunology

基　　金：天津市自然科学基金资助（06YFJRIC06900）

摘　　要：目的通过同源重组法构建LuxS基因缺失的变形链球菌（Streptococcus mutans）突变株。方法运用基因同源重组方法将红霉素抗性基因（Eym‘）连接到PCR扩增LuxS基因两端区域产生的2个基因片段之间，并共同插入到pUC19载体的多克隆位点中，构建出带红霉素抗性标志的缺失突变载体pUCluxKO。将突变载体转化到含完整LuxS基因的突变受体变形链球菌标准株中，红霉素筛选出LuxS基因缺失的变形链球菌突变株，并经PCR、生物荧光检测及DNA测序鉴定。结果构建的突变载体经限制性内切核酸酶酶切分析显示，产生的条带与设计结果完全一致。PCR方法扩增突变株LuxS和Eym＇基因显示，LuxS基因已被完整敲除掉，经生物荧光检测，突变株不能诱导哈氏弧菌（Vibrio hazy／）BB170的生物发光，说明不能产生信号分子AI-2（autoinducer-2）。DNA测序证实筛选得到了LaxS基因缺失的变形链球菌突变株。连续传代培养后证实，变形链球菌LaxS基因突变株具有良好的稳定性。结论成功构建出LaxS基因缺失的变形链球菌突变株，为研究LaxS基因对变形链球菌致龋毒力的影响奠定了基础。Objective To knock out the entire LuxS gene of Streptococcus mutans UA159 strain via homologous recombination and construct a LuxS-deleted mutant strain of S. mutans. Methods The erythromycin resistance gene （Eym＇） was inserted between the two DNA fragments located in the upper and downstream of LuxS gene that had been amplified by PCR. Then the two DNA fragments along with the inserted Eymr were engineered into pUC19 plasmid to construct the recombination plasmid pUCluxKO. Electrotransformation of S. mutans cells with pUCluxKO-mutant resulted in the isolation of erythromycin resistant S. mutans transformants, which was then subjected to polymerase chain reaction, Vibrio harveyi BB170 luminescence bioassay and sequencing analysis. Results Restriction endonuclease analysis showed that pUCluxKO- mutant vector had been successfully recombined. The deletion of LuxS of S. mutans mutants was confirmed by PCR with primers specific for the genes of LuxS and the erythromycin resistance. S. mutans mutant could not induce bioluminescence, indicating the mutant had been successfully recombine＆ The constructed Chinese S. mutans showed good stability after 20 generations of cultivation. Conclusion The S. mutans gene allelic exchange plasmid is constructed correctively and a LuxS-negative mutant of S. mutans has been constructed, which can be helpful for further study of the role of LuxS in the pathogenesis of S. mutans.

关 键 词：变形链球菌 群体感应 LUXS基因 AI-2 突变株 

分 类 号：R686[医药卫生—骨科学]