表皮葡萄球菌luxS基因敲除重组质粒的构建

目的构建1个含有luxS基因上、下游片段、抗卡那霉素基因的重组克隆质粒，用以敲除表皮葡萄球菌luxS基因。方法检索GenBank获得luxS基因序列以设计引物，以生物膜阳性表皮葡萄球菌基因组DNA为模板，采用高保真PCR扩增得到包含luxS基因上游片段、完整的luxS基因和luxS基因下游片段的长片段DNA；以pEASYT4质粒为模板扩增得到抗卡那霉素基因，再用内侧引物分别扩增luxS基因上、下游序列，按luxS基因上游片段＋抗卡那霉素基因＋luxS基因下游片段的顺序重组连接，转化JMl09感受态细胞，通过卡那霉素筛选、酶切分析和PCR一测序验证luxS基因敲除的重组克隆载体。结果经酶切后电泳验证重组质粒中各目的片段插入无误，PCR检测结果证明重组质粒luxS基因缺失，测序结果显示碱基无错配，含目的基因的重组质粒菌株在含卡那霉素培养平板内正常生长，证明插入的抗卡那霉素基因表达良好。结论表皮葡萄球菌luxS基因敲除重组质粒构建成功，为后续luxS基因缺陷株的构建奠定了基础。

Construction of the cloned plasmid of luxS gene knockout of Staphylococcus epidermidis

Objective To construct a cloned plasmid containing the upstream and downstream fragments of the luxS gene and kanamycin resistance gene for the following homologous recombination knockout experiments on luxS gene of S. epidermidis. Methods The luxS gene sequence was obtained by retrieving the GenBank for designing primer, the long fragment DNA containing the upstream and downstream fragments of the luxS gene, the complete luwS gene were obtained by PCR amplification using biofilm-positive strains genomic DNA as template, and the kanamycin resistance gene was amplify by PCR using pEASY T4 plasmid as template. Then the upstream and downstream sequence of the luxS gene was amplified by PCR using the inner primer. The DNA fragments were connected in the correct sequence of the upstream fragment of luxS gene the kanamycin resistance gene and the downstream of luxS gene were transformed into JM109 competent cells. Then recombinant cloning vector was obtained by kanamycin screening for the knockout of the lu~z＇S gene and validated through enzyme electrophoresis and PCR-sequencing. Results Each fragment was inserted correctly by enzyme electrophoresis. The luxS gene deletion of the recombinant plasmid was proved by PCR. Sequence analysis showed that the base groups do not mismatch. The recombinant plasmid strains containing target gene grew normally in kanamycin culture plate. It proved that the inserted kanamycin resistance gene was expressed well. Conclusion The recombinant plasmid of the luxS gene knockout in S. epidermidis was successfully constructed. This study laid a solid foundation for construction of the luxS gene knockout S. epidermidis.