Author/Authors :
Hasanzadeh Haghighi, Faria Antimicrobial Resistance Research Center - Mashhad University of Medical Sciences - Mashhad, Iran , Aryan, Ehsan Antimicrobial Resistance Research Center - Mashhad University of Medical Sciences - Mashhad, Iran , Derakhshan, Mohammad Antimicrobial Resistance Research Center - Mashhad University of Medical Sciences - Mashhad, Iran , Meshkat, Zahra Antimicrobial Resistance Research Center - Mashhad University of Medical Sciences - Mashhad, Iran , Gholoobi, Aida Dept. of Modern Sciences and Technologies - School of Medicine - Mashhad University of Medical Sciences - Mashhad, Iran
Abstract :
Tuberculosis (TB) remains a major cause of death around
the world. Bacillus Calmette Guérin (BCG) is the only vaccine used in TB prevention
that has a protective effect in children, but its effectiveness declines in adults. Design
and development of new vaccines is the most effective way against TB.
The aim of this study was to design and construct a DNA vaccine encoding mtb32C
and mpt51 fusion genes of Mycobacterium tuberculosis.
Methods: First, mpt51 fragment was amplified by PCR method. The pcDNA3.1+/
mtb32C plasmid was transformed into E. coli JM109 and then extracted. The mpt51
gene and pcDNA3.1+/mtb32C plasmid were both digested with EcoRI and BamHI restriction
enzymes followed by ligation of mpt51 fragment into the digested vector. The
recombinant plasmid containing mtb32C and mpt51 was subsequently transformed
into competent E. coli TOP10 strain. The clones were confirmed by colony-PCR,
restriction enzyme digestion and sequencing.
Results: Using agarose gel electrophoresis, a 926 bp fragment corresponded to
mpt51 was observed. Digestion of the vector pcDNa3.1+/mtb32C and mpt51 gene was
confirmed by electrophoresis. Then, the pcDNA3.1+/mtb32C plasmid was extracted.
Sequencing results confirmed the accuracy of the desired plasmid.
Conclusion: In this study, we constructed a cloning vector encoding mtb32C/mpt51
gene of M. tuberculosis. The eukaryotic expression of this vector can be confirmed in
future studies. It can be considered as a DNA vaccine in animal models later. Successful
cloning provides a basis for the development of new DNA vaccines against TB.
Keywords :
Cloning , Genetic vectors , Antigens , Mycobacterium tuberculosis