中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2008年
3期
198-202
,共5页
于丹妮%韩福胜%韩玉植%陈杰
于丹妮%韓福勝%韓玉植%陳傑
우단니%한복성%한옥식%진걸
变形链球菌%群体感应%LuxS基因%AI-2%突变株
變形鏈毬菌%群體感應%LuxS基因%AI-2%突變株
변형련구균%군체감응%LuxS기인%AI-2%돌변주
Streptococcus mutans%Quorum sensing%JuxS gene%A1-2%Mutant
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(Eymr)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pUCl9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Eymr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 harveyi)BBl70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LuxS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LuxS基因突变株具有良好的稳定性.结论 成功构建出LuxS基因缺失的变形链球菌突变株,为研究LuxS基因对变形链球菌致龋毒力的影响奠定了基础.
目的 通過同源重組法構建LuxS基因缺失的變形鏈毬菌(Streptococcus mutans)突變株.方法 運用基因同源重組方法將紅黴素抗性基因(Eymr)連接到PCR擴增LuxS基因兩耑區域產生的2箇基因片段之間,併共同插入到pUCl9載體的多剋隆位點中,構建齣帶紅黴素抗性標誌的缺失突變載體pUCluxKO.將突變載體轉化到含完整LuxS基因的突變受體變形鏈毬菌標準株中,紅黴素篩選齣LuxS基因缺失的變形鏈毬菌突變株,併經PCR、生物熒光檢測及DNA測序鑒定.結果 構建的突變載體經限製性內切覈痠酶酶切分析顯示,產生的條帶與設計結果完全一緻.PCR方法擴增突變株LuxS和Eymr基因顯示,LuxS基因已被完整敲除掉,經生物熒光檢測,突變株不能誘導哈氏弧菌(Vibrio harveyi)BBl70的生物髮光,說明不能產生信號分子AI-2(autoinducer-2).DNA測序證實篩選得到瞭LuxS基因缺失的變形鏈毬菌突變株.連續傳代培養後證實,變形鏈毬菌LuxS基因突變株具有良好的穩定性.結論 成功構建齣LuxS基因缺失的變形鏈毬菌突變株,為研究LuxS基因對變形鏈毬菌緻齲毒力的影響奠定瞭基礎.
목적 통과동원중조법구건LuxS기인결실적변형련구균(Streptococcus mutans)돌변주.방법 운용기인동원중조방법장홍매소항성기인(Eymr)련접도PCR확증LuxS기인량단구역산생적2개기인편단지간,병공동삽입도pUCl9재체적다극륭위점중,구건출대홍매소항성표지적결실돌변재체pUCluxKO.장돌변재체전화도함완정LuxS기인적돌변수체변형련구균표준주중,홍매소사선출LuxS기인결실적변형련구균돌변주,병경PCR、생물형광검측급DNA측서감정.결과 구건적돌변재체경한제성내절핵산매매절분석현시,산생적조대여설계결과완전일치.PCR방법확증돌변주LuxS화Eymr기인현시,LuxS기인이피완정고제도,경생물형광검측,돌변주불능유도합씨호균(Vibrio harveyi)BBl70적생물발광,설명불능산생신호분자AI-2(autoinducer-2).DNA측서증실사선득도료LuxS기인결실적변형련구균돌변주.련속전대배양후증실,변형련구균LuxS기인돌변주구유량호적은정성.결론 성공구건출LuxS기인결실적변형련구균돌변주,위연구LuxS기인대변형련구균치우독력적영향전정료기출.
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(Eymr)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 pUCl9 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 BBl70 luminescence bioassay and sequencing analysis.Results Restriction endonuclease analysis showed that pUCluxKOmutant vector had been successfully recombined.The deletion of LuxS of S. mutans mutants was confirmed bv 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 recombined.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.
