关键词: 变异链球菌, htrA基因, HtrA蛋白, Cre/loxP位点特异性重组系统, 基因缺失

Abstract: 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. Electrotransformation of S. mutans cells with pIB△htrA-Km resulted in isolation of kanamycin resistant S. mutans transformants. 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 temperature, resulting in a markerless mutant strain carrying a deletion at the htrA loci, which was verified by PCR and DNA sequencing. Result: 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 loci. 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.

Key words: Streptococcus mutans, htrA gene, HtrA protein, Cre/loxP site-specific recombination system, gene deletion