3.1. Mice and Parasites
Female BALB/c mice (six to eight weeks), obtained from the Pasture Institute of Iran, were maintained in the animal house during the experiments. All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985). The L. major strain MRHO/IR/75/ER parasites were kept in a virulent state through continuous passage in BALB/c mice. The L. major promastigotes were cultured at 26°C in RPMI-1640 (Gibco, USA) medium supplemented with 10% heat inactivated FCS (Gibco, USA), 10mM HEPES, 2mM L-glutamine, and 40 μg/mL gentamycin.
3.2. Recombinant SODB1 Preparation
Preparation of rSODB1 has been described previously (9). Briefly, the coding region of Leishmania major SODB1 was amplified by specific primers, which contained specific sequences for digestion by NdeI (Thermo Fisher Scientific, Lithuania) and XhoI (Thermo Fisher Scientific, Lithuania), as well as the initiation codon. The purified PCR product was cloned directly in pGEM-T easy T vector TA cloning (Invitrogen, USA) and then ligated in the NdeI-XhoI insertion site of expression vector pET22-b (Novagen, USA). The construct was transformed to E. coli BL21 (DE3) and hexa-His-tag fusion protein was expressed by induction with 0.2 mM isopropyl-b-D-thiogalactoside (IPTG). Inclusion bodies were isolated from lysed bacteria and after solubilization in urea (5 M) containing buffer, recombinant protein containing the histidine-tag was affinity purified using IDA-chelating Sepharose (Amersham Pharmacia Biotech AB, Sweden). Purification of recombinant protein was assessed by SDS-PAGE analysis. Purified recombinant protein was gradually dialyzed against guanidine chloride-containing PBS buffer. The concentration of guanidine chloride was 0.5 mM at the end of the experiment.
3.3. Soluble Leishmania Antigen
The Soluble Leishmania Antigen (SLA) was prepared from the stationary phase of L. major promastigotes. The cells were harvested after centrifugation at 4000 × g (Sorvall RC-5 centrifuge, HB-4 swinging bucket rotor) at 4°C for 15 minutes. The pellet was washed with PBS (pH 7.2) and re-suspended in the same buffer to a density of 106 cells/mL. Sonication was used to lyse the cells (four pulses of 1 min using an MSE sonicator with an intensity setting of 15 mm amplitude). The lysate was centrifuged at 17000 × g for 15 minutes at 4°C, and the supernatant was used as SLA. The protein concentrations of the rSODB1 and SLA were measured by the Bradford method.
3.4. Immunization and Challenge with Parasite
Twenty-four mice were categorized to three groups. The first group (immunized group) was immunized with 50 μg of the rSODB1 protein in Complete Freund’s adjuvant (rSODB1+CFA) in a total volume of 100 μL. Control groups received complete Freund’s adjuvant (control group I) or PBS (control group II). Two other subcutaneous booster injections were given in the back of the mice at two-week intervals using incomplete FA instead of complete FA. Three weeks after the second booster injection, the animals were challenged with 1×106L. major meta-cyclic promastigotes (suspended in 50 μL PBS), injected in the right footpad. The progress of infection was monitored for eight weeks by determining the foot-pad swelling, using a metric caliper.
3.5. Cytokine Assay
Exactly before and eight weeks after the challenge, four mice from each group were sacrificed. The splenocytes were cultured using RPMI-1640 medium, supplemented with 10% FCS (Gibco), 2 mM glutamine, 10 mM HEPES, and 40 μg/mL gentamycin. Cells were seeded in triplicates in 96 well plates (Nunc, Denmark) at 2×105 cells/well and stimulated with either rSODB1 (10 μg/mL), or SLA antigen (10μg/mL), or cultured with medium alone. Phytohemagglutinin (PHA), at concentrations of 2 μg/mL, was used in all experiments as a positive control. After 72 hours, supernatants were collected and tested for the IFN-γ, IL-5, and IL-10 production, using the sandwich-based ELISA kits (R&D, Minneapolis, MN, USA), according to the manufacturer’s instructions. The lower detection limits of IFN-γ, IL-5, and IL-10 were 2, 7 and 5 pg/mL, respectively. All experiments were performed in triplicates for four mice.
3.6. Statistical Analysis
The differences in lesions size and cytokine levels were determined by one-way analysis of variance (ANOVA). In case of significant P value, multiple comparisons (Tukey test) were used to compare the means of different treated groups. All statistical analyses were performed using the SPSS software (version 11.5). Differences were considered statistically significant when P < 0.05.
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