The Study Of MAGE-1 Peptide Immunological Effect In Vitro

Operation therapy,radiation therapy and chemotherapy are the principle therapeutics for the malignant tumors currently. Operation therapy is the most commonly used methods to deal with cancer and can achieve a satisfying effect in many malignant tumors which arise locally early. But the operation therapies cannot get a satisfying result or even cannot be performed in the malignant tumors which have diffused such as leukemia and tumors inadvanced stage and in the malignant tumors which infiltrate important organs and transferred delitescencely to distant place. Radiation therapy and chemotherapy can also kill and wound the normal tissue and produce great side effects. Resistance to the drugs can also limit the application of chemotherapy. The currently therapies cannot be satisfying in the treatment for the malignant tumors.As medical oncologists become more familiar with immune-based strategies, the cancer vaccines may play a more prominent role in treating patients with various types of malignancies.The biological behaviors of malignant tumors have been recognized more deeply with the development of molecular biology and immunology in the past few years. Malignant tumors are one kind of gene-associated diseases and tumor cells are not only similar and homological but heterological with normal tissue. Tumor cells accumulate DNA mutations during neoplastic transformation that lead to the production of altered or novel antigens(tumour associated antigen or tumour specific antigen,TAA or TSA ) that are not found in normal adult tissue, and which represent potential targets for selective recognition by the host immune system.The cells of malignant tumors can activate a specific immune response to the tumour cell. And the immune responses include humoral and cell-mediated immune responses,the later is more important.T cells play a great role in cytoimmunity.Human cytotoxic T cells are able to recognize 9- to 14-mer antigenic peptides expressed within the major histocompatibility complex (MHC) on the surface of all cells. When appropriately activated, Tcells can detect specific TAAs within the MHC and initiate targeted, immune-mediated cell killing. The ideal TAA is specific to, or overexpressed on, the surface of cancer cells. The fundamental goal of cancer immunotherapy is to induce a targeted immune response against cancer cells. In 1991,MAGE-1 was first identified from the melanoma cell line MZ2-MEL and recognized by host cytotoxic T-lymphocytes (CTL).From then on, many genes were found, and most were encoded by multigene families on chromosome X, MAGE-1is an ideal TAA(tumor associated antigen)for the development of immunotherapy . MAGE-1 genes are expressed in testis, but not in any other normal somatic tissues or cells, including melanocytes. In contrast, these genes are expressed in a proportion of many different types of cancers, including breast cancer, lung cancer, and ovarian cancer, etc. This expression pattern imply that these gene products are tumor-specific antigens, The T-cell responses to MAGE antigens are typically investigated by the screening of overlapping peptide panels with CD8+ or CD4+T-cells from peripheral blood. Many HLA-restricted T-cell epitopes have been identified this way.We can use cDNA encoding full-length protein and also can use partial cDNA encoding specific epitope. The use of peptide vaccines is an attractive approach because of the ease of synthesis and manipulation of these vaccines.Moreover,in contrast to whole-gene vaccines,peptide vaccines can induce immune responses against specific Ag epitopes while avoiding the interference of nonrelevant Ag epitopes.Consequently,such vaccines lend themselves to in-depth studies of immunological mechanisms far more readily than do DNA vaccines encoding entire genes. Much work on different links of gene immunity has been done to enhance the immune effect of DNA vaccines.In antigen modifying,many research groups are interested in selecting and combining the predominant epitope and in removing the suppressed epitope.We can analyse the sequence of Ag epitope and recognize its immunologic function exactly along with the development of gene technology and immunology.Because MHC class I-restricted CTLs have a direct lytic effect on tumor cells,most efforts have focused on the identification of peptide epitopes capable of stimulating these types of responses. In view of this,one obvious way to improve vaccines designed to induce antitumor CTLs is to include in these vaccines MHC class II-restricted epitopes.In agreement with this assessment , we selected a polyepitope ,which contained three high-affinity CTL epitopes .Mononuclear cells,which harvesting from human peripheral blood we differentiated into DCs by rhGM-CSF and rhIL-4.After charging with nonapeptide of MAGE1,DCs and T cells were mixed with SMMC-7721 cells.MTT chromatometry was used to examine the inhibition ability of CTLs. The DCs activated by MAGE-1 nonapeptide can induce specific inhibition against SMMC- 7721 cells.In the second part, we obtained the DNA fragment encoding MAGE-1127-169 from human genome by PCR.The sequenced gene was subcloned into expression vector,then purified from transformed E.coli DH5αusing Ni-NTA affinity chromatography column and assessed by Western blotting with anti-his monoclonal antibody.Lymphocytes from mice were tested in vitro for specific proliferation responses to the MAGE-1127-169 peptide by 3H-TdR incorporation.The result showed that A 6×his-MAGE-1127-169 fusion protein was expressed in E.coli DH5αand mainly located in inclusion bodies.Western blotting with anti-His monoclonal antibody identified the fusion protein.In the present of various concentration of MAGE-1127-169 peptide,lymphocytes exhibited increase of proliferation response after 7-9 days , and it could induce the CTL effects of T-cells. Concluded that the MAGE-1127-169 peptide was successfully expressed in E.coli and could promote antigen-specific response in vitro.Our research made a helpful exploration into expression and purification of short peptide and suggested that MAGE-1127-169 peptide could act as a lymphocyte epitope processing strong immunogenicity .