| ObjectiveTo predict the H-2Kd-restricted CTL epitopes of tumor-associated antigen ROR1, modify the epitope peptide with thymosin alpha 1 to synthesize ROR1-Tal tumor vaccine and investigate the anti-tumor effect of the vaccine.MethodsEpitope peptides were predicted with super motif combined and quantitative motif by BIMAS and SYFPEITHI. The candidate epitope peptides were synthesized and purified. Analysis of binding ability of the candidate epitope peptides with H-2Kd molecules was carried out by peptide binding assay. We synthesized the most promising epitope with thymosin alpha 1 into a coupling peptide RORl-Tal. ELISA method was used to check IFN-y and IL-12 level. BALB/c mice were immunized with ROR1-Tal 3 times, once a week. Mouse breast cancer cells 4T1 were implanted into the mice at day 7 after first immunization. Mice were sacrificed 3 weeks after the last immunization and tumor weight was observed.ResultsThe predicted four candidate epitope peptides ROR1 716-724 (DCPPRMYSL), ROR1 269-277 (SNPMILMRL), ROR1 788-796 (NYMFPSQGI) and ROR1 591-599 (DFLHIAIQI) had high affinities with H-2Kd, the FI values were more than 1.5 when peptide concentrations are more than 30μg/ml. ELISA results showed that RORl-Tal could stimulate spleen lymphocyte producing IFN-y and IL-12. ROR1-Tal can inhibit the growth of tumor in vivo.ConclusionsROR1 591-599 (DFLHIAIQI) is a CTL epitope of the breast cancer associated antigen ROR1 presented by H-2Kd. RORl-Tal peptide vaccine has biological activity in vitro and exerts immunoprotective effect in vivo. This research provides an experimental basis for further study of the immunologic mechanism of RORl-Tal peptide vaccine. |
