利用框内缺失法构建变异链球菌srtA基因缺失株

发布时间：2018-07-10 17:54

本文选题：变异链球菌 + srtA基因缺失　；参考：《华西口腔医学杂志》2017年06期

【摘要】：目的利用框内缺失法,通过IFDC2基因盒及融合聚合酶链反应(PCR)、同源重组技术构建变异链球菌UA159菌株srt A基因缺失株。方法 PCR扩增变异链球菌UA159菌株srt A基因上下游同源片段及IFDC2基因盒,将这些片段连接、转化入UA159,替换srt A基因同源片段,筛选、鉴定红霉素抗性克隆;PCR扩增、连接UA159 srt A基因上下游同源片段,将连接片段转化入前述红霉素抗性克隆中替换IFDC2同源片段,筛选、鉴定抗苯丙氨酸类似物p-chloro-phenylalanine(p-Cl-Phe)克隆。结果 PCR、琼脂糖凝胶电泳及DNA测序结果均证明srt A基因编码区被完全删除,上下游片段无缝连接,成功构建UA159 srt A基因缺失株。结论本研究成功构建了无标记的变异链球菌UA159 srt A基因缺失株,为进一步研究该基因在生物膜中的作用及其调控机制奠定了基础。
[Abstract]:Objective to construct srt A gene deletion strain of Streptococcus mutans UA159 strain by using IFDC2 gene box and fusion polymerase chain reaction (PCR). Methods the upstream and downstream homologous fragments of srt A gene and IFDC2 gene cassette of Streptococcus mutans UA159 strain were amplified by PCR. The fragments were ligated into UA159 and transformed into UA159. The upstream and downstream homologous fragments of UA159 srt A gene were ligated and transformed into the aforementioned erythromycin resistant clones to replace the IFDC2 homologous fragments. P-chloro-phenylalanine (p-Cl-Phe) clones resistant to phenylalanine analogue were screened and identified. Results the results of PCR, agarose gel electrophoresis and DNA sequencing showed that the coding region of srt A gene was completely deleted, and the upstream and downstream fragments were seamlessly connected, and UA159 srt A gene deletion strain was successfully constructed. Conclusion this study successfully constructed an unlabeled srt A gene deletion strain of Streptococcus mutans UA159, which laid a foundation for further study on the role of the gene in biofilm and its regulatory mechanism.
【作者单位】：口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心四川大学华西口腔医院牙体牙髓病科;
【基金】：国家自然科学基金(81570974) 四川省科技计划项目基金(2015JY0260)~~
【分类号】：R780.2

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