Faria Hasanzadeh Haghighi - Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Ehsan Aryan - Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Aida Gholoobi - Department of Modern Sciences and Technologies, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Hosna Zare - Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Zahra Meshkat - Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran and Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Tuberculosis (TB) is the leading cause of death by infectious diseases worldwide, and especially prevalent in developing countries. Several vaccines against TB have been developed, recently. The aim of the present study was to design and construct a cloning vector encoding Mycobacterium tuberculosis (MTB) mpt51 gene. Methods: DNA was extracted from MTB H37Rv strain. Gene-specific primers were designed using Gene Runner software and the mpt51 gene was amplified by PCR. The amplified fragment and pcDNA3.1(+) cloning vector were both digested with restriction enzymes, the mpt51 fragment was ligated into the vector, and the Escherichia coli (E. coli) TOP10 strain were transformed by the recombinant plasmid. Positive clones were identified by colony PCR, restriction enzyme digestion, and DNA sequencing. Results: The mpt51 gene was successfully cloned into pcDNA3.1(+). A 6400 bp band for the pcDNA3.1(+)/mpt51 recombinant plasmid and a 926 bp band for mpt51 were observed by colony PCR, and restriction enzyme digestion on agarose gels. The DNA sequence was 100% homologous with the mpt51 fragment of H37Rv in GenBank. Conclusions: In the current study, the mpt51 gene of MTB was correctly cloned into pcDNA3.1(+). The expression of this recombinant vector can be studied in eukaryotic cells. Moreover, it is possible to determine the efficacy of this vector as a DNA vaccine candidate, and to test its protective function compared to BCG in animal models in future.  

Molecular Cloning, MPT51 antigen, gene, .