变形链球菌荧光报告株的构建和鉴定

The construction and identification of the ldh-luc reporter strain of Streptococcus mutans and the reporter strain

目的探讨变形链球菌(S.mutans)荧光素酶(luc)基因报告株构建方法,拟为探讨抗菌剂对多菌种生物膜中S.mutans的作用提供研究基础。方法将S.mutans UA159乳酸脱氢酶(ldh)基因及其上游部分约1100000片段克隆至自杀质粒pFW5-luc的多克隆位点,构建重组质粒,并经酶切和测序证实。采用自然转化的方法,实现重组自杀质粒和S.mutans UA159同源序列的单次交换。结果经过聚合酶链反应(PCR)和测序分析,筛选出具有报告活性的S.mutans ldh-luc基因荧光报告株。结论成功构建了S.mutans ldh-luc基因荧光报告株。

Objective To construct the ldh-luc reporter strain of Streptococcus mutans （S. mutans） and identity the reporter strain. Methods Approximately 1 100 000 of sequence upstream of the ldh start codon from S.mutans genome were amplified and cloned to the suicide vector pFWS-luc. Confirmed constructs were transformed into S.mutans and integrated via single crossover homologous recombination. The expected integration was confirmed in antibiotic-resistant clones by PCR and sequencing. Results The ldh-luc reporter strain of S.mutans was selected and confirmed by PCR and sequencing analysis of multiple clones for similar reporter activity. Conclusion An ldh-luc reporter strain of S.mutans was constructed successfully.