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3.1 Expression of wE7, uE7 and IL-12 proteins in Cos7
cells

To study the expression
of wE7, uE7 and IL-12 proteins in cos-7 cells, crude cell lysates were
separated by electrophoresis on SDS-polyacrylamide gel and were identified
using of mouse anti-E7 and mouse anti-IL12 antibodies. Western blot analysis of
cell lysates showed proteins with two size of approximately 35 KDa and 82KDa for
wE7/ uE7 and IL-12 recombinant proteins, respectively. These results confirmed
expression of the proteins as it migrated according to its corresponding size (Fig.1).

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Fig.1. Detection of
recombinant proteins in Cos7 cells by Western blot analysis. Lane 1, a 82KDa
band was observed in pcDNA3.1+-IL12 transfected cells using mouse anti-IL12,
Lane 2, molecular weight protein marker (Sinaclon, Iran); lane 3, negative
results in pcDNA3.1+ transfected cells and lane 4 and 5, two recombinant bands
with size of approximately 35KDa was detected in pcDNA3.1+- wE7/ uE7
transfected cells using mouse anti-E7. The blots were developed with ECL.

3.2 LDH Cytolytic responses

LDH release results
show significantly enhanced CTLs responses against wE7&IL12 and uE7&IL12.
As shown in figure 2, lymphocytes from the mice vacci­nated with uE7&IL12 (67±
2.6)  wE7&IL12 (67± 2.6)  had a significantly increased specific
cytolytic activity at an E/T ratio of 50:1 than those vacci­nated with PBS (12.1%±0.70%),
pcDNA3.1+ (19.1%±0.99%), pcDNA3.1+wE7 (37.4%±2.09%), or
pcDNA3.1+uE7 (41.3%±2.2%) (P< 0.05). No statistically significant difference in cytolytic activity was found by LDH assay between the wE7&HSP70 and uE7&HSP70 groups. At E/T ratios of 20:1 there was no significantly difference between all groups.         Fig. 2. Quantitative measurement of LDH release in immunized mice. Data were collected from LDH release results at the different E/T ratios expressed as percent cytotoxicity ± SD. The data shown here are from 3 independent experiments in triplicate. 3.3 T cell proliferation response MTT Results showed an increased proliferation index or SI in wE7&IL12 and uE7&IL12 vaccinated groups, as compared to others groups: PBS, pcDNA3.1+, pcDNA3.1+-wE7 or pcDNA3.1+-uE7 (p<0.05) (Fig. 3). Moreover, no statistically significant difference in SI by MTT assay was found between the uE7&HSP70 and wE7&HSP70 groups.         Fig.3. Splenocyte proliferation levels after in vitro re-stimulation with specific antigen. The results show that the stimulation index values of the uE7&HSP70 and wE7&HSP70 vaccinated mice increases significantly compared to other groups (p <0.05).   3.4 Secretion levels of cytokines IFN-? level was significantly increased in the wE7&IL12 and uE7&IL12 vaccinated mice (470.33±66 and 436.4±55 respectively) as compared with other groups (P<0.01) (Fig. 4).               Fig. 4. Determination of the production of Th1 cytokines by splenocytes of immunized mice. The levels of statistical significance for differences between test groups were determined using ANOVA. Each sample was examined in triplicate (p <0.05).   3.5 In vivo tumor challenge assays As shown in figure 7, mice treated with wE7&IL12 and uE7&IL12 vaccines underwent to significant decrease in tumor volumes, as compared to mice treated with PBS, pcDNA3.1+, pcDNA3.1+-wE7, or pcDNA3.1+-uE7 (P<0.05). The results indicate that vaccination with wE7&IL12 and uE7&IL12 induces a more efficiently therapeutic antitumor effect than vaccination in the control groups. Fig. 7. Assessment of therapeutic vaccine on tumor size between the 5th and 7th weeks after the tumor inoculation. Results show that the differences in wE7&IL12 and uE7&IL12 vaccinated groups was statistically significant (p<0.05). The tumor growing curve of the pcDNA3.1+ wE7&IL12 and uE7&IL12 group was the lowest among the mice in the seven groups. Line and scatter plot graphs depicting the tumor volume (in mm3) are presented. The data presented are a representation of 2 independent experiments.                

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