利用Cre/loxP系统构建无标记的htrA基因缺失的变链菌突变株

Construction of Unmarked HtrA-Deficient Mutant of Streptococcus Mutans with Cre/loxP System

目的:利用Cre/loxP位点特异性重组系统构建口腔变异链球菌htrA基因缺陷株,并删除选择标记。方法:PCR扩增变链菌的htrA基因片段并克隆到pGEM-T-EasyTA载体。随后插入卡那霉素(Km)抗性基因盒(loxP-Km-loxP)并替换htrA基因的部分序列,使htrA基因失活,构建出htrA基因缺失的同源重组载体pIB△htrA-Km。将该质粒电转化变链菌标准株,抗生素筛选出发生同源重组的菌株,再用含cre基因的热敏质粒pCrePA电转化,删除Km抗性基因。在限制性温度下培养,消除质粒pCrePA,获得无标记的变链菌htrA基因缺陷株,经PCR及DNA测序鉴定。结果:PCR及DNA测序分析证明htrA基因的部分序列及Km抗性基因均被删除,该部位只留下一个loxP位点。结论:成功构建出了变链菌htrA基因缺失突变株,并删除了抗性标记,为进一步研究htrA基因缺失对变链菌致龋毒力的影响奠定了基础。

Objective： To construct the Streptococcus mutans （S.mutans） htrA-deficient mutant and to remove the antibiotic resistance marker with the Cre/loxP site-specific recombination system.Methods： The DNA fragment containing htrA was amplified by PCR and cloned into the pGEM-T-Easy TA cloning vector.Then,a kanamycin resistance cassette（loxP-Km-loxP）was inserted into this recombinant plasmid and replaced a part of the htrA gene,yielding homologous recombination plasmid pIB△htrA-Km.The electrotransformation of S.mutans cells with pIB△htrA-Km resulted in the isolation of kanamycin resistant S.mutans transformant.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 the nonpermissive temperature,resulting in a markerless mutant strain carrying a deletion at the htrA loci,which was verified by PCR and DNA sequencing.Results： The deletion of a part of htrA and the kanamycin resistance gene was confirmed by PCR analysis and DNA sequencing.There was a loxP at this locus.Conclusion： A htrA-deficient mutant of S.mutans was constructed and the antibiotic resistance marker was deleted successfully,which can help to further study the role of htrA in the pathogenesis of S.mutans.