In this study, a vehicle vector using cryptic plasmids was constructed for gene transfer in Helicobacter pylori. pHP51 (3.9 kb) and pHP489 (1.2 kb) were selected for constructing vectors from cryptic plasmid of H. pylori isolates in Korea. The
HindⅢ-digested DNA fragment (1.2 kb) of pHP489 and 1.6 kb DNA fragment of pHP51 were ligated with a kanamycin resistance gene (aph3'-Ⅲ) from C. jejuni to produce the recombinant plasmids pHP489K and pHP51K, respectively. Transformation
frequency
of
pHP51K by electroporation was low. But pHP489k could be effectively transformed into various H. pylori strains. In order to design an intermdiate vehicle vector for gene transfer into H. pylori, pBlueHP489k was prepared by recloning pHP489K DNA
into
pBluescript and pTZ19R vector. This vector permitted the DNA fragment containing pHP489 sequence, aph3'-Ⅲ, and cloned DNA to be cut and self-ligated in the SacⅠsite after cloning. ureB gene were inserted into pBlueHP489K, resulting in
pBlueHP489K/AB.
The DNA fragment containing pHP489, kanamycin resistance gene (aph3'-Ⅲ), and urease structural gene was cut away from pBlueHP489K/AB and self-ligated to generate pHP489K/AB pHP489K/AB made urease-negative H. pylori strains restore their urease
activity. By this experiment, pBlueHP489K was confirmed to be the vehicle system for transferring H. pylori genes.
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