Plates were then washed, air-dried and spots were counted using an ELISPOT reader (CTL Co.). To reveal roles of CD4+and Selleckchem CHIR99021 CD8+ T cells in the immune response, splenocytes were depleted of CD4+ or CD8+ T cells by using corresponding antibody (Miltenyi Biotec Inc.) before ELISPOT assays. Cytotoxicity assay Splenocytes were harvested from three mice per group one week after the final vaccination, and then incubated with irradiated Renca-vIII(+)cells(EGFRvIII transfected Renca cells).
Five days later, T cells were harvested and purified from the cultures using lymphocyte separating buffer. These T cells were used as CTL effector cells and co-cultured with target cells renca-vIII(+)cells at various effector/target ratios for 8 h at 37°C. Values were expressed as the percentages of surviving Renca-vIII(+)cells cultured with effector cells. Renca cells which were not transfected with EGFRvIII served as control. Tumor find more challenge Thirty BALB/c mice were divided into three group(10 mice pre group), and immunized with fusion protein, HBcAg and PBS. After five times of immunization, antibody titers of mice immunized with fusion protein reached 2 × 105. Then all mice were challenged with 1.5 × 105 Renca-III(+) cells in the left flank. Tumor growth was measured and volumes were calculated according to the formula V = (a2·b2·c2)/6, where V represents tumor volume and a, b, and c were
perpendicular diameters of the tumor. After observation, mice were killed, and tumors were weighted. Statistical analyses All data were expressed as means
± SD. Comparisons between individual data points were performed by Student’s t -test. Data for quantitation were evaluated by analysis of variance (ANOVA). p < 0.05 was considered statistically significant. Results Construction of recombinant expression plasmids The PCR product and recombinant plasmid were detected by restriction analysis (Figures 2, 3 and 4) and then sequenced. The results showed that the compound gene Pep-3, cloning plasmid Pep3-HBcAg/pGEMEX-1, and expression plasmid Pep3-HBcAg/pET-28a (+) were successfully constructed. Figure 2 Identification of PCR product. lane1: PCR product of Pep-3; lane2: DNA Marker of 200 bp. Figure 3 Identification of plasmid Pep3-HBcAg/pGEMEX-1. lane1: cloning plasmid Pep3-HBcAg/pGEMEX-1 digested with EcoR I and Xho I; lane 2: pep3-HBcAg/pGEMEX-1 clonidine plasmid without digestion; lane 3:λDNA/Hind III marker(23.13 Kb, 9.414 Kb, 6.557 Kb, 4.371 Kb, 2.082 Kb, 0.564 Kb, 0.125 Kb); lanel 4: 100 bp DNA Ladder. Figure 4 Identification of plasmid pep3-HBcAg/pET-28a (+). Lanel1: λDNA/Hind III marker; lanel 2: 100 bp DNA Ladder; lane 3: recombinant expression plasmid pep3-HBcAg/pET-28a (+) digested with EcoR I and Sal I; lane 4: pep3-HBcAg/pET28a (+) plasmid without digestion. Expression and purification of the fusion protein To obtain the fusion protein, the engineering strains E. coli BL21 (DE3) were cultured in 2 × YT with 0.