The Application Of Bioinformatics In The Redication Of Cytotoxic T Lymphocyte Epitopes Of Tumor Antigen MAGE-A Sub-family And Research Of Immunological Activity Induced By Epitopes

It has been shown that tumor antigen, especially the tumor specific antigen (TSA), which can induce tumor-specific cytotoxic T lymphocyte (CTL) and damage tumor cell, is the major mechanism of tumor vaccine. The melanoma antigen (MAGE) was the first tumor specific antigens to be discovered. MAGE-3 is one mumber of MAGE gene family, which expresses in most malignant tumors but not in normal tissue except for testis and placenta, has been used as the ideal target for tumor immunotherapy. MAGE-n was a new member of MAGE gene family, which was firstly reported by our research group (GeneBanK, Locus No.AF443295). It is highly homologous to MAGE-A subfamily genes and has been demonstrated closely asscociated with Hepatocellular carcinoma (HCC) in our previous study.In this study, the HLA-A2 restricted CTL epitopes of tumor antigen MAGE-A sub-family were predicted by SYFPEITHI prediction method combined with the polynomial method. The amino acid sequence and the candidate CTL epitopes of the member of MAGE-A sub-family were analyzedby DNAstar software. Using epitope reconstruction, We investigated the immunological effects of CTL epitope candidates of MAGE-3 and MAGE-n. The research was divided into two parts as following:Experiment 1: Prediction and analysis of HLA-A2-restricted CTL epitopes derived from the tumor antigen MAGE-A sub-familyObjiective To predict the HLA-A2-restricted CTL epitopes of tumor antigen MAGE-A sub-family, and provide appropriate CTL epitope for tumor immunotherapy. Methods The CTL epitopes of tumor antigen MAGE-A sub-family were predicted by SYFPEITHI prediction method combined with the polynomial method. The amino acid sequence and the candidate CTL epitopes of the member of MAGE-A sub-family were analyzed by DNAstar software. Results Fifty HLA-A2-restricted CTL epitope candidates(nonamers) derived from the tumor antigen MAGE-A sub-family were selected, and some epitopes were highly similar or completely identical. Conclusion The combination of SYFPEITHI prediction method and polynomial method can improve the prediction efficiency and accuracy. Analysing the homology of CTL epitopes and finding common epitopes can develop some promising antigen peptide for tumor vaccines.Experiment 2: The anti-tumor effect of CTL epitopes ofMAGE-3 and MAGE-n in vitroObjiective To study the immunological activity of the HLA-A2-restricted CTL candidate epitopes derived from tumor antigen MAGE-3 and MAGE-n by epitope reconstrution methods, ELISPOT and specific cytotoxicity assay. Methods The MAGE-3 and MAGE-n candidate HLA-A2 restricted CTLepitopes were synthesized with solid phase strategies, purified with reverse phase HPLC and identified with mass spectrometry.Expression of the MAGE-3 and MAGE-n gene in tumor cell lines were detected by RT-PCR. The HHCC cells transfected with MAGE-3 gene was established by liposome transfection. Peptide-specific CTLs were obtained from the peripheral blood mononuclear cells (PBMC) of HLA-A2 positive healthy donors by multiple stimulations with peptide-pulsed T2 cells. The ELISPOT assay was used to determine the frequency of T cells responding to antigen peptide by secretion of IFN-γ. The capability of CTLs recognizing and lysing HLA-A2+ MAGE-3+and MAGE-n+ tumor cells was detected with lactate dehydrogenase (LDH) release assay. Results KATO-Ⅲ cells expressed MAGE-3 and MAGE-n gene. MCF-7 cells expressed MAGE-3 gene, and HHCC cells expressed MAGE-n gene. The HHCC-MAGE-3 cell line was established by liposome transfection. The results of IFN-γ ELISPOT demonstrated that the association of MAGE-3 and MAGE-n epitope could induce a higher number specific T cells than other groups (p<0.05).All HCC cell lines used in this study were HLA-A2 positive cells. The average IFN-γ spot numbers of responding lymphocytes induced by T2 pulsed with single epitope HLA-A2 peptide were much higher than that of responding lymphocytes induced by T2 cells without peptide (P<0.05). The ratio of CD3+ T cells of responding lymphocytes increased to 97%, and the ratio of CD8+ T cells increased to 68% after 24-days culture. The cytotoxicity measured by LDH release assay showed the CTLs stimulated with two epitopes peptide exhibited specific lysis of tumor cell lines expressing both MAGE-3 and MAGE-n, the lysis rate are 61%, 68.8%, 62.3%,70.2%,75.8% separately , but CTL stimulated with single...