First step in developing an epitope-based vaccine is to predict peptide binding to the major histocompatibility complex (MHC) molecules. We performed computational analysis of unique available EgA31 sequence to locate appropriate antigenic propensity positions. T-cell epitopes with best binding affinity values of < 50% inhibitory concentration were selected using different available servers (Propred and IEDB). Peptides with 100% population coverage were selected. A DNA fragment corresponding to the furin linker enriched in Golgi apparatus was inserted sequentially between each epitope sequences in a synthetic DNA in order to cleave the chimeric protein into four separated peptides. Subsequently, the synthetic DNA was cloned into the pGEX4T-1 and pEGFP-N1 vectors and GST-ChEgA31 was expressed in E. coli strain BL21-DE3. The recombinant protein was detected by western blotting using an HRP-conjugated polyclonal anti-GST antibody. Fusion protein purified by affinity chromatography was used to raise antisera in rabbits. Results in agar gel immunodiffusion assay indicated induction of specific antibodies against multiepitope antigen in the tested rabbits. Cytokine assay was carried out in C57Bl/6 mice and the levels of cytokines were analyzed by sandwich ELISA. Interestingly, production of specific IFN-gamma was prominently higher in mice immunized with GST-ChEgA31 and pEGFP-ChEgA31 (650-1300 pg/ml) compared to control groups. No difference was observed in the level of IL-10 and IL-4 in immunized and GST control group. Challenge study with 500 live protoscolices of Echinococcus granulosus on immunized mice demonstrated protectivity level (50-60%). Based on our results, it appeared that the chimeric protein in the study was able to stimulate T-helper cell-1 (Th1) development and high level of cell mediated immunity in mice.

• MeSH
• antigeny helmintové imunologie   MeSH
• cytokiny genetika   metabolismus   MeSH
• DNA helmintů MeSH
• Echinococcus granulosus imunologie   MeSH
• ELISA MeSH
• epitopy T-lymfocytární genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• králíci MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• regulace genové exprese imunologie   MeSH
• rekombinantní proteiny MeSH
• Th1 buňky imunologie   MeSH
• vakcíny imunologie   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH