| Over the last few years, several human genes that code tumor antigens recognized by autologous CTL have been isolated and the peptide epitopes derived from these tumor antigens have been further identified to serve as targets for CTL in the context of HLA class I molecules. Among these tumor antigen genes, MAGE gene family has been shown to be expressed in a variety of human malignant tumors, but not in normal tissue except for the testis. The 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.Hepatocellular carcinoma (HCC) is one of the commonest malignant diseases in China and some other parts of Asia. Although encouraging advances have been made in the past few decades, there are still many difficulties in treating advanced-stage patients and in preventing recurrence and metastasis. The immunotherapy is always considered promising despite the fact that it is still a supplementary method in clinical practice. In the Chinese population, the frequent of HLA-A2 types in HCC patients is higherthan that of other HLA-I phenotype. It is very valuable to study whether the MAGE-n antigen peptide presented by HLA-A2 could induce primary, peptide-specific CTL against to HCC cells.In this study, the HLA-A2 restricted CTL epitope of MAGE-n were identified by the methods of combined prediction , epitope reconstruction and immunological assay. The ability of MAGE-n epitope QLVFGIEVV specific CTLs in suppression the growth of human hepatocellular carcinoma was studied in SCID mice. It is proved that the epitope QLVFGIEVV (MAGE-n 159-167) has high affinity to HLA-A2 and could induce specific CTL from the PBMC of healthy HLA-A2 positive donors in vitro, farthermore these CTLs lysed HLA-A2 positive HCC cells expressing MAGE-n in this study. The immunological activity specific to HCC in vivo was demonstrated by prevention and therapy experiments of xenograft human HCC tumor on SCID mice. There are three parts reported as follows in this essay:Experiment 1; Elptope predictionObjiective To predict the HLA-A2-restricted CTL epitopes and biological characteristic of tumor antigen MAGE-n. Methods The HLA-A020l restricted CTL epitopes of tumor antigen MAGE-n were predicted by SYFPEITHI prediction methods combined with the polynomial methods. The biological characteristic of MAGE-n encoding protein was predicted with ANTHEPROT V5.0 software. Results Five HLA-A2-restricted CTL epitope candidates (nonamers) derived from the tumor antigen MAGE-n were selected by prediction method SYFPEITHI combined with the polynomial method. The hydrophobicity, Hydrophilicity, Antigenicity, Helical memeberanous regions, pHi and Potential cleavage site of signal peptide ofMAGE-n were obtained by ANTHEPROT V5.0 software analysis. Conclusion The combination of SYFPEITHI prediction method and polynomial method can improve the prediction efficiency and accuracy.Experiment 2;lmmunologlcal characteristic off predictedepltopesObjiective To study the binding affinity and immunological activity of the HLA-A2-restricted CTL candidate epitopes derived from tumor antigen MAGE-n with epitope reconstrution methods, ELISPOT and specific cytotoxicity assay. Methods The MAGE-n candidate HLA-A2 restricted CTL epitopes were synthesized with solid phase strategies, purified with reverse phase HPLC and identified with mass spectrometry. The binding affinity and binding stability of synthesized peptides were studied by cellular competition-based HLA-A2 peptide binding assay, T2 peptide stabilization assay and peptide-MHC complex dissociation assay. Expression of the MAGE-n gene in HCC cell lines was detected by RT-PCR. The SMMC-7721 cell line transfected MAGE-n gene was established by liposome transfection. The ELISPOT assay was established for determination the frequency of T cells capable of responding to antigen pe...
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