Construction of Novel Plasmid Vectors for Gene Knockout in Helicobacter pylori
Construction of Novel Plasmid Vectors for Gene Knockout in Helicobacter pylori
复制标题
幽门螺杆菌基因敲除新型质粒载体的构建
DOI：
10.1007/s00284-016-1140-7
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发表时间：
2016-12-01
影响因子：
2.6
通讯作者：
Li, Boqing
中科院分区：
文献类型：
--
作者：
Ji, Xiaofei;Zhao, Huilin;Li, Boqing
幽门螺杆菌感染在各种胃十二指肠疾病的病因中起着重要作用。但H. pylori不清楚。更多的潜在致病因素有待进一步发现和研究。本研究利用两种载体在H. pylori的质粒pLYL03的骨架设计。重组质粒pLYL03上的基因，减少冗余序列。将pLYL 03上的红霉素抗性基因替换为aphA或catGC，得到卡那霉素抗性质粒pSJHK或氯霉素抗性质粒pSJHC。pSJHK和pSJHC的大小分别为4371和3949 bp。以质粒pSJHK和pSJHC为基础，构建了以hp0169和hp0788为靶点的双交换重组基因敲除质粒，并通过电穿孔法获得缺失突变体。结果表明，质粒pSJHK和pSJHC是有效的基因缺失在H。pylori的转化效率，pSJHC的转化效率略高于pSJHK。这些质粒为进一步研究H.幽门。通过改变耐药基因开发的衍生质粒也将为研究其他细菌中的功能基因提供有价值的工具。
Helicobacter pylori infection plays an important role in the etiology of various gastroduodenal diseases. However, pathogenic mechanism of H. pylori is not clear. More potential pathogenic factors need to be further discovered and studied. In this study, two vectors for generating double-crossover recombination gene knockout plasmids in H. pylori were designed based on the backbone of plasmid pLYL03. Genes on plasmid pLYL03 were rearranged, and the redundant sequences were reduced. Erythromycin-resistant gene on pLYL03 was replaced by aphA or catGC to generate the kanamycin-resistant plasmid pSJHK or the chloramphenicol-resistant plasmid pSJHC. The sizes of pSJHK and pSJHC are 4371 and 3949 bp, respectively. Based on plasmids pSJHK and pSJHC, double-crossover recombination gene knockout plasmids targeting hp0169 and hp0788 were constructed, and deletion mutants were achieved by electroporation of the gene-targeting plasmids. The results indicated that plasmids pSJHK and pSJHC are efficient for gene deletion in H. pylori, and the transformation efficiency of pSJHC is slightly higher than pSJHK. These plasmids provide convenient genetic tools for further research of novel pathogenic factors in H. pylori. Derivative plasmids developed by changing antibiotic-resistant genes would also provide valuable tools for the study of functional genes in other bacteria.