Design and Construction of a Eukaryotic Cloning Vector Encoding the mpt51 Gene of .

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 gene.

METHODS

DNA was extracted from MTB H37Rv strain. Gene-specific primers were designed using Gene Runner software and the gene was amplified by PCR. The amplified fragment and pcDNA3.1(+) cloning vector were both digested with restriction enzymes, the fragment was ligated into the vector, and the TOP10 strain were transformed by the recombinant plasmid. Positive clones were identified by colony PCR, restriction enzyme digestion, and DNA sequencing.

RESULTS

The gene was successfully cloned into pcDNA3.1(+). A 6400 bp band for the pcDNA3.1(+)/ recombinant plasmid and a 926 bp band for were observed by colony PCR, and restriction enzyme digestion on agarose gels. The DNA sequence was 100% homologous with the fragment of H37Rv in GenBank.

CONCLUSION

In the current study, the 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.