| Immunotherapy is one of the adjuvant treatment to malignant tumor, theeffective antitumor immunity depends on activation of both arms of the immune system, humoral and cellular, especially CD8+T-cells, which can inhibit tumor growth and eradicate cancer cells. In addition, CD4+ T-cells can generate and maintain potent antitumor immunity.The identification of tumor specific antigen is the key to induction of specific antitumor immunity. A series of tumor-specific antigens had been identified since three tumor antigens were first found by Boon. Among the MAGE gene family, MAGE-3 (MAGE-A3) is one of the tumor-specific antigens, it expresses in a high proportion of melanomas and in many human malignant tumor including hepatocellular carcinoma, head and neck squamous cell carcinoma, testicular germ cell tumor, breast cancer, no-small cell lung cancer, gastric carcinoma, colorectal carcinoma, etc. MAGE-3 doesn' t express in normal tissues, with exception of testis and placenta. So MAGE-3 is regarded as ideal target for specific antitumor immune responses.Cancer vaccines, including peptide-based vaccines, protein vaccines, and DNA vaccines, could be one of the ideal therapies because those vaccines are inducing cellular and humoral immune responses and targeting tumor-specific antigens. The use of protein vaccines has much more clinical value as it provids a wide range of multiple identified or unidentified MHC class I and II epitopes which could induce specific antitumor immune responses.In this study, the recombinant expression plasmid pGEX-4T-2/MAGE-3 was constructed by ligating MAGE-3 gene, which was amplified by RT-PCR from human placenta and confirmed by sequencing, and the pGEX-4T-2 vector. The recombinant plasmids were transformed into BL-21 Escherichia coli. The expression of GST-MAGE-3 was induced with IPTG, checked by SDS-PAGE and Western blot, purified with Glutathione Sepharose 4B. The recombinant pcDNAS. 1+/MAGE-3 mammalian expression plasmid were constructed successfully, and a B16-hMAGE-3 cell line expressed the MAGE-3 were consequently set up. Then the specific antitumor immune responses induced by autologous peripherial blood mononuclear cells (PBMCs) from patient with gastric cancer pulsed with antigen GST-MAGE-3 in vitro were investigated. Finally, the specific antitumor immune responses including CD8+ and CD4+ T cells induced by lymphocytes from murine spleen loaded with antigen GST-MAGE-3 in vivo were also investigated.Section One Construction of GST-MAGE-3 prokaryotic expression plasmid and its expression in Escherichia coliObjective: To construct GST-MAGE-3 prokaryotic expression plasmid , express the GST-MAGE-3 protein in Escherichia coli and purify the protein. Methods: The recombinant expression plasmid pGEX-MAGE~3 was constructed by ligating MAGE-3 gene, which was amplified by RT-PCR and confirmed by sequencing, and the pGEX-4T~2 vector. The recombinant plasmid was transformed into BL-21 E. coli.. The expression of GST-MAGE-3 was induced with IPTG. After purification with Glutathione Sepharose 4B, the purity of the protein was more than 90% , and 3mg GST-MAGE-3 was obtained from 100 ml BL-21 lysate. Results: The recombinant pGEX-4T-2/MAGE-3 plasmid was constructed correctly by sequencing and the expression of GST-MAGE-3 fusion protein in BL-21 was confirmed. Conclusion: The fusion protein GST-MAGE-3 was obtained successfully.Section Two Peripherial Blood Mononuclear Cells from patient with gastric cancer pulsed with GST-MAGE-3 antigen induce specific antitumor immune responseObjective: To investigate specific antitumor immune responses induced by autologous peripherial blood mononuclear cells (PBMCs) from patient with gastric cancer pulsed with antigen GST-MAGE-3 in vitro. Methods: Peripherial blood mononuclear cells (PBMCs) obtained from gastric carcinoma patients were regarded as antigen presenting cells after pulsed with GST-MAGE-3, then cocultured with autologous lymphocytes, after that, the lymphocytes were collected, lymphocyte proliferative abil...
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