Tuberculosis (TB) kills about two million people each year, becoming one of the world's leading infectious causes of death among young people and adults. One-third of the world's population is infected with TB. Each year, nearly 2 million people die of TB, despite the availability of inexpensive treatments that are effective in up to 95% of cases. In particular, pulmonary tuberculosis agent is the Mycobacterium tuberculosis. To date, the prevention has been done with BCG vaccine, which presents a TB resistance among 50% and 80%, showing little impact in TB control in the world. In this work we proposed the development of a prototype of subunit vaccine against TB through the construction of a recombinant chimera. Epitope sequences of M. tuberculosis Hsp65 were chosen based on earlier works (Mustafá et al., 1999; Charo et al., 2001) to form a chimerical protein with five epitope sequences. The DNA encoding the epitope sequences was obtained by the synthetic gene assembly through PCR reaction. The PCR product was cloned in pGEM-T Easy vector. A positive clone was sequenced and subcloned in the expression vector pET29b. The chimerical protein was then expressed in E.coli BL21(DE3) cells induced by 0.2mM IPTG and grown up for 3 hours at 37ºC, under agitation. The recombinant protein obtained in the soluble form was purified by affinity chromatography using a Ni-NTA resin. In this step, all the materials were treated with NaOH (0.5-1M) to avoid lipopolysaccharide contamination. The recombinant protein has been used in the immunological trials with BALB/c mouse to evaluate immune response in different times. In addition, lymphoproliferation trials have been conducted with animal's spleen cells.