Design and construction of fusion genes hspX and tb10.4 from Mycobacterium tuberculosis in a cloning vector
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Date
2017-03-31
0000-0002-4032-7599 Journal Title
Journal ISSN
Volume Title
Publisher
Brieflands
Abstract
Introduction: Tuberculosis (TB) is the most important infectious disease and is one of the most common causes of death in the world especially in developing countries. TB is caused by infection with Mycobacterium tuberculosis. Designing and construction new vaccines against Mycobacterium tuberculosis are the only effective way to prevent and control the disease. The aim of this study was to design and construct a cloning vector encoding hspX and tb10.4 fusion genes of Mycobacterium tuberculosis.
Materials and Methods: At first, tb10.4 fragment was amplified by PCR method and it was digested with restriction enzymes and was cloned into the plasmid pcDNA3.1 +. Then, hspX fragment was amplified by PCR method and it was digested by HindIII and BamHI restriction enzymes. The recombinant plasmid pcDNA3.1 + / tb10.4 also digested with the same enzymes and hspX was subcloned into the recombinant vector. This construct was transformed into the Escherichia coli strain TOP10. The confirming the clones were performed by colony PCR, restriction enzyme digestion and sequencing methods.
Results: PCR and enzymatic digestion products were run on the agarose gel and tb10.4 and hspX genes were observed 291bp and 435bp, respectively. In addition, results of DNA sequencing showed 100% homology with hspX and tb10.4 genes of Mycobacterium tuberculosis H37Rv strain recorded in GenBank.
Conclusion: In this study, hspX and tb10.4 genes of Mycobacterium tuberculosis were cloned into pcDNA3.1 + vector correctly. This vector can use as a DNA vaccine to induce immune system responses in animal models in future studies.