中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2011年
2期
102-106
,共5页
于丹妮%张文娟%彭诚%韩育植%任志明
于丹妮%張文娟%彭誠%韓育植%任誌明
우단니%장문연%팽성%한육식%임지명
链球菌,变异%基因缺失%Cre-loxP系统
鏈毬菌,變異%基因缺失%Cre-loxP繫統
련구균,변이%기인결실%Cre-loxP계통
Streptococcus mutans%Gene deletion%Cre-loxP system
目的 利用以Cre-loxP为基础的位点特异性重组系统Cre-loxP*构建变形链球菌的htrA和clpP双基因缺失突变株,为利用该系统构建无标记的多基因缺陷株奠定基础.方法 将htrA基因克隆到pGEM-T-Easy TA载体,并插入卡那霉素抗性基因愈(lox71-Km-lox66)使htrA基因失活,获得htrA基因删除载体pGEM-T-△htrA/Km.用相同方法构建带有大观霉索抗性标记的clpP基因删除载体pGEM-T-△clpP/Sp.用pGEM-T-△htrA/Km转化变形链球菌标准株,筛选出发生同源重组的菌株.用cre表达质粒pCrePA转化同源重组菌株,删除抗性基因.由于pCrePA的热敏复制子,改变温度培养可消除pCrePA,得到无标记的htrA基因缺失突变株MS△htrA,并经聚合酶链反应(PCR)及DNA测序鉴定.用同样的方法在MS△htrA中删除clpP及大观霉索抗性标记,获得无标记的htrA和clpP双基因缺失突变株MS△htrA-△clpP.结果 PCR及DNA测序鉴定结果表明,htrA和clpP基因及抗性标记已被删除,无标记的htrA和clpP双基因缺失突变株成功构建.结论 利用位点特异性重组系统Cre-loxP*初步构建出变形链球菌的无标记htrA和clpP双基因缺失突变株,为将该系统应用于构建口腔变形链球菌无标记的多基因缺陷株提供了实验依据.
目的 利用以Cre-loxP為基礎的位點特異性重組繫統Cre-loxP*構建變形鏈毬菌的htrA和clpP雙基因缺失突變株,為利用該繫統構建無標記的多基因缺陷株奠定基礎.方法 將htrA基因剋隆到pGEM-T-Easy TA載體,併插入卡那黴素抗性基因愈(lox71-Km-lox66)使htrA基因失活,穫得htrA基因刪除載體pGEM-T-△htrA/Km.用相同方法構建帶有大觀黴索抗性標記的clpP基因刪除載體pGEM-T-△clpP/Sp.用pGEM-T-△htrA/Km轉化變形鏈毬菌標準株,篩選齣髮生同源重組的菌株.用cre錶達質粒pCrePA轉化同源重組菌株,刪除抗性基因.由于pCrePA的熱敏複製子,改變溫度培養可消除pCrePA,得到無標記的htrA基因缺失突變株MS△htrA,併經聚閤酶鏈反應(PCR)及DNA測序鑒定.用同樣的方法在MS△htrA中刪除clpP及大觀黴索抗性標記,穫得無標記的htrA和clpP雙基因缺失突變株MS△htrA-△clpP.結果 PCR及DNA測序鑒定結果錶明,htrA和clpP基因及抗性標記已被刪除,無標記的htrA和clpP雙基因缺失突變株成功構建.結論 利用位點特異性重組繫統Cre-loxP*初步構建齣變形鏈毬菌的無標記htrA和clpP雙基因缺失突變株,為將該繫統應用于構建口腔變形鏈毬菌無標記的多基因缺陷株提供瞭實驗依據.
목적 이용이Cre-loxP위기출적위점특이성중조계통Cre-loxP*구건변형련구균적htrA화clpP쌍기인결실돌변주,위이용해계통구건무표기적다기인결함주전정기출.방법 장htrA기인극륭도pGEM-T-Easy TA재체,병삽입잡나매소항성기인유(lox71-Km-lox66)사htrA기인실활,획득htrA기인산제재체pGEM-T-△htrA/Km.용상동방법구건대유대관매색항성표기적clpP기인산제재체pGEM-T-△clpP/Sp.용pGEM-T-△htrA/Km전화변형련구균표준주,사선출발생동원중조적균주.용cre표체질립pCrePA전화동원중조균주,산제항성기인.유우pCrePA적열민복제자,개변온도배양가소제pCrePA,득도무표기적htrA기인결실돌변주MS△htrA,병경취합매련반응(PCR)급DNA측서감정.용동양적방법재MS△htrA중산제clpP급대관매색항성표기,획득무표기적htrA화clpP쌍기인결실돌변주MS△htrA-△clpP.결과 PCR급DNA측서감정결과표명,htrA화clpP기인급항성표기이피산제,무표기적htrA화clpP쌍기인결실돌변주성공구건.결론 이용위점특이성중조계통Cre-loxP*초보구건출변형련구균적무표기htrA화clpP쌍기인결실돌변주,위장해계통응용우구건구강변형련구균무표기적다기인결함주제공료실험의거.
Objective To construct double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans(Sm) and to remove the antibiotic resistance markers with the Cre-loxP*site-specific recombination system. Methods The htrA gene was cloned into the pGEM-T-Easy TA cloning vector and then inactivated via the insertion of a kanamycin resistance cassette(lox71-Km-lox66), yielding pGEM-T-△htrA/Km for deleting the htrA gene. Using the same method, the pGEM-T-△clpP/Sp was constructed for deleting the clpP gene. Following the transformation of pGEM-T-△htrA/Km in Sm, the homologous recombination event was selected. One such mutant was transformed with a cre expression plasmid (pCrePA). The kanamycin resistance gene was then excised. The pCrePA was then easily eliminated at nonpermissive temperatures, resulting in a mutant strain (MS △ htrA) carrying a deletion at the htrA loci without a selectable marker. This mutant was verified by PCR and DNA sequencing. Then, the clpP and spectinomycin resistance gene were deleted from MS △htrA, yielding markerless mutant strain lacking clpP and htrA. Results The deletion of htrA, clpP and antibiotic resistance markers were confirmed by PCR analysis and DNA sequencing. Conclusions A mutant of Sm was constructed successfully which contained a deletion of the htrA and clpP gene without selectable marker. The Cre-loxP*system can be applied to Sm, which provides experimental evidence for generating markerless multiple gene deletion mutants.
