Cloning and sequencing of M. tb gene Rv0378 for making subunit based DNA vaccine

Mycobacterium tuberculosis has infected one third of the world's population and 1.5 million people die each year due to tuberculosis. The research was conducted to make clones of M. tb gene Rv0378 during January-November, 2015. Gene Rv0378 has a gene length 222bp and was amplified using proper sequence specific primers. The size and quality of DNA fragments were determined by agarose gel electrophoresis. PCR product was then ligated in Vector PtZ57R/T (T/A vector) in order to be transformed into competent alpha-DH5 (E. coli) cells. Ampicillin positive clones were selected from the plates, introduced in the autoclaved test tubes with 2 to 3 ml. SOB broth was then placed in a shaker incubator for overnight at 37ºC. Next day turbidity was clearly observed in the test tubes indicating the culture was ready to use for plasmid extraction. DNA was extracted by using Genejet Plasmid extraction miniprep Kit by Thermo scientific. Size of the extracted construct was about >3000bp (other impurities of proteins and salts are washed away in EZ-Spin columns). In next step it will be cloned into pND14 (A mammalian expression vector) to make candidate vaccine and will be tested for its efficacy against M. tb.