Iranian journal of microbiology · 2013
Cloning of Vibrio cholerae outer membrane protein W in Pichia pastoris.
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Alizadeh Javad, Ranjbar Reza, Kamali Mehdi, Farhadi Nima, Davari Amin, Sadeghifard Nourkhoda
Аннотация
BACKGROUND AND OBJECTIVE: The outer membrane protein W (ompW) of Vibrio cholerae is involved in stimulating the immune response via induction of protective immunity. It also plays an important role in bacterial pathogenesis by increasing the adaptability of pathogenic strains. In this study we aimed to clone V. cholerae ompW gene in the strain X-33 of Pichia pastoris.
MATERIALS AND METHODS: A gene encoding ompW was cloned into the Ppicza vector downstream of alcohol oxidase promoter. Then recombinant vector was transformed into the genome of the strain X-33 of P. pastoris. After growth of zeocin-resistant transformants, clones were selected and subsequently confirmed for cloning by PCR enzymatic digestion and sequencing.
RESULTS: PCR, enzymatic digestion and sequencing showed that the ompW gene was correctly cloned into P. pastoris genome.
CONCLUSION: Results of our study showed that the methylotrophic yeast P. pastoris can be considered as an appropriate host instead of mammalian and prokaryotic systems for cloning of ompW. As far as data show, this is the first time that ompW of V. cholera is cloned into the methylotrophic P. pastoris.
MATERIALS AND METHODS: A gene encoding ompW was cloned into the Ppicza vector downstream of alcohol oxidase promoter. Then recombinant vector was transformed into the genome of the strain X-33 of P. pastoris. After growth of zeocin-resistant transformants, clones were selected and subsequently confirmed for cloning by PCR enzymatic digestion and sequencing.
RESULTS: PCR, enzymatic digestion and sequencing showed that the ompW gene was correctly cloned into P. pastoris genome.
CONCLUSION: Results of our study showed that the methylotrophic yeast P. pastoris can be considered as an appropriate host instead of mammalian and prokaryotic systems for cloning of ompW. As far as data show, this is the first time that ompW of V. cholera is cloned into the methylotrophic P. pastoris.