SDS-PAGE analysis demonstrated that recombinant fusion protein was efficiently and inducibly expressed in inclusion body form and could dissolve in 6 M urea. The molecular weight of the fusion protein was shown to be approximately 15.4 kD as expected. According to the results of SDS-PAGE and gel image analysis, the purified fusion protein accounted 92% of totle protein (Figure 5). Western blot analysis demonstrated that the fusion protein had specific antigenicity against anti-EGFRvIII antibody (Figure 6). Figure 5 SDS-PAGE analysis of recombinant selleck kinase inhibitor protein. Lane 1: protein molecular weight marker; Lane2: negative control: recombinant plasmid Pep3-HBcAg/pET28a
(+) transformed E. coli BL21 cells not induced by IPTG; Lane 3: HBcAg/pET28a (+) transformed E. coli BL21 cells induced by IPTG Lane 4, 5: supernatant and sediment of recombinant plasmid Pep3-HBcAg/pET28a (+) induced by IPTG; Lane 6: purified recombinant fusion
protein. Figure 6 Western blot analysis. Lane 1: Western blot of pET28a (+); Lane 2: Western blot of EGFRvIII-HBcAg fusion protein using EGFRvIII-specific monoclonal antibody; Lane 3: protein marker. Immunization assay of fusion protein To investigate whether the EGFRvIII-HBcAg fusion protein could induce humoral immune response, the tail EVP4593 mw vein serum samples were collected on day 0, 14, 21, 28, 35, 42 and 48, and the antibody titers against the fusion protein were tested by ELISA (Figure 7). Figure 7 Time course of immunization response. Mice immunized with fusion protein produced specific antibody responses, which increased significantly from week 5 and peaked at week 7. However, no obvious antibody response was detected in mice immunized with HBcAg or PBS. Induction of specific CTL response ELISPOT assay was carried out to determine the frequency of lymphocytes secreting Florfenicol IFN- γ. The
number of IFN- γ secreting cells was very low in mice immunized with HBcAg or PBS alone, whereas mTOR cancer vaccination with fusion protein induced a high frequency of IFN- γ -secreting cells (p < 0.05) (Figure 8). To identify which cell populations were involved in the IFN- γ production, the CD4- or CD8-depleted splenocytes from mice immunized with fusion protein were detected. The depletion of CD4+ T cells could completely abrogate IFN- γ production by the harvested splenocytes, but the depletion of CD8+T cells had no influence on the number of ELISPOTs (Figure 9). This finding suggest that CD4+ T cells, but not CD8+ T cells, play an important role in anti-tumor activity of fusion protein. Figure 8 Frenquency of IFN-γ-secreting cells in splenocytes from mice innunized with fusion protein was much higher than that in HBcAg or PBS. Figure 9 Frequency of IFN-γ-secreting cells in CD4- depleted splenocytes was dramatically lower than CD8- depleted splenocytes. Furthermore, the cytotoxic activity of splenocytes from mice was examined (Figure 10, 11).