Designing and Construction Pcdna3.1 Vector Encoding Cfp10 Gene of Mycobacterium tuberculosis

BACKGROUND Pathogenic mycobacteria are a major cause of human morbidity and mortality. Mycobacterium tuberculosis is an etiological agent of human tuberculosis (TB). Designing new vaccines, including DNA vaccines, may be a useful strategy for preventing TB. OBJECTIVES The purpose of this study was to design and construct an eukaryotic expression vector containing M. tuberculosis. MATERIALS AND METHODS Genomic DNA of M. tuberculosis H37Rv cultured on Lowenstein Jensen medium was extracted, and cfp10 was amplified by PCR. After digesting the PCR product and the plasmid, the cfp10 fragment was ligated into the vector pcDNA3.1 (+). Correct insertion was confirmed by colony PCR, restriction enzyme digestion, and sequencing. RESULTS Electrophoresis of the PCR product on gel showed a 303-bp target fragment. Colony PCR, restriction enzyme digestion, and Sequencing methods confirmed the accuracy of the gene cloning. Colony PCR and restriction enzyme digestion confirmed the cloning. CONCLUSIONS Cloning of cfp10 of M. tuberculosis into an eukaryotic expression vector was performed successfully. We propose this recombinant plasmid for inducing immunity in animal models in future studies. This recombinant vector can also be used in the construction of fusion proteins.