应用Cre-loxP＊系统构建无标记的变形链球菌双基因缺陷株  

Application of Cre-loxP＊ system in constructing the markerless double-gene-deletion strain in Streptococcus mutans

作　　者：于丹妮[1] 张文娟[1] 彭诚[1] 韩育植[1] 任志明[1] 

机构地区：[1]天津医科大学第二医院口腔科,300211

出　　处：《中华口腔医学杂志》2011年第2期102-106,共5页Chinese Journal of Stomatology

摘　　要：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.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.

关 键 词：链球菌 变异 基因缺失 Cre-loxP系统 

分 类 号：Q78[生物学—分子生物学]