Cloning of Vibrio cholerae outer membrane protein W in Pichia pastoris.
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.


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.


CONCLUSIONS
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.
Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Javad
J
Alizadeh
Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Reza
R
Ranjbar
Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Mehdi
M
Kamali
Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Nima
N
Farhadi
Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
Amin
A
Davari
Clinical Microbiology Research Center, Ilam University of Medical Sciences, Ilam, Iran.
Nourkhoda
N
Sadeghifard
PCR; Pichia pastoris; Sequencing; Vibrio cholerae; cloning; outer membrane protein W