Construction of Novel Plasmid Vectors for Gene Knockout in Helicobacter pylori.

Abstract

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.