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Helicobacter pylori와 대장균의 Shuttle Vector 개발
조명제,이우곤,이상룡,김경희,안영숙,김성희,김현주,류복덕,최여정,윤영혜,백승철,전영석,이광호 경상대학교 유전공학연구소 1997 遺傳工學硏究所報 Vol.16 No.-
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-digestedDNA fragment(1.2kb) of pHP489 and 1.6kb 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 SacⅠ site after cloning. ureA and 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 pBlueHP489K/AB. pBlueHP489K/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.