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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pEASY-T4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JM109感受态细胞,通过卡那霉素筛选、酶切分析和PCR-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。
Objective To construct a recombinant cloning plasmid containing upstream and downstream fragments of luxS gene and kanamycin resistance gene to knock out the luxS gene of Staphylococcus epidermidis. Methods GenBank was used to obtain the luxS gene sequence to design primers. Using biofilm-positive Staphylococcus epidermidis genomic DNA as a template, long fragment DNA containing upstream fragment of luxS gene, complete luxS gene and downstream fragment of luxS gene was amplified by high-fidelity PCR. The anti-kanamycin gene was amplified by using the plasmid pEASY-T4 as a template, and then the upstream and downstream sequences of the luxS gene were amplified by the inner primers. The sequence of the fragment upstream of the luxS gene plus the fragment of the anti-kanamycin gene + downstream of the luxS gene Recombinant ligation, transformation of JM109 competent cells, kanamycin screening, digestion analysis and PCR-sequencing to verify luxS gene knockout recombinant cloning vector. Results After digestion electrophoresis, it was verified that the inserted fragments were inserted correctly. PCR results showed that the luxS gene of the recombinant plasmid was deleted. The sequencing results showed that the recombined plasmids had no mismatch with the recombinant plasmids containing the target gene. Growth within the normal, proved inserted kanamycin gene expression is good. Conclusion The luxS gene knockout recombinant plasmid of Staphylococcus epidermidis was successfully constructed, which laid the foundation for the construction of the following luxS gene defective strains.