Lb liquid medium

The vaccine pVAXhsp65 was constructed from the pVAX vector (Invitrogen, Carlsbad, CA, USA). This plasmid was digested with BamHI and NotI (Gibco BRL, Gaithersburg, MD, USA) and then the M. leprae hsp65 gene and the CMV intron A were inserted. DH5α E. coli transformed with pVAX or pVAXhsp65 were cultured in LB liquid medium (Gibco BRL, Gaithersburg, MD, USA) containing kanamycin (100 μg/ml). The plasmids were purified using the Concert High Purity Maxiprep System (Gibco BRL, Gaithersburg, MD, USA). Plasmid concentrations were determined by spectrophotometry at λ = 260 and 280 nm by using the Gene Quant II apparatus (Pharmacia Biotech, Buckinghamshire, UK).

The DNA-hsp65 vaccine was constructed by inserting the gene that expresses the M. leprae hsp65 protein into the cloning site of the pVAX plasmid (Invitrogen^®, Carlsbad, CA, USA) previously digested with the restriction enzymes Bam HI and Not I (Gibco BRL, Gaithersburg, MD, USA). The empty vector DNA-pVAX, without the hsp65 gene, was used as a control. DNA-hsp65 and DNA-pVAX used for vaccinations were produced in Escherichia coli transformed with the respective plasmids and cultivated in LB liquid medium (Gibco BRL) containing 100 µg/mL of ampicillin. The plasmids were then purified using the Concert High Purity Maxiprep System (Gibco BRL). The Gene Quant II apparatus (Pharmacia Biotech, Buckinghamshire, UK) was used to evaluate the concentration of plasmids in the samples.