| Tumor vaccine, as an important approach for tumor biological therapies, has being more and more attended, and exploring an efficient, broad-spectrum tumor vaccine has become a hotspot in the areas of tumor prevention and trerapy in recent years. Tumor antigen, especially the tumor specific antigen (TSA), which can generate tumor-specific cytotoxic T lymphocyte (CTL) and damage tumor cell, is the major mechanism of tumor vaccine. It is difficult to find a potential specific tumor antigen and make it presented efficiently in the design of tumor vaccine. Furthermore, the limitation of MHC and the polymorph of tumor cells are the major factors of the limited region and/or the low efficiency of tumor vaccine. Heat shock proteins (HSPs), a highly conserved protein in vivo (or isolating cells), participate in the folding, transport and assembling of proteins, so named "molecular chaperone". The expression of HSPs can be induced by the stresses such as heat and lack of glucose. On the other hand, HSPs play an important role in the processing and presenting ofantigens, which could conjugate proteins or peptieds and improve their antigenicity. Tumor protein vaccine can be processed and presented by immune system, so it can be used in patients with different HLA without HLA limitation, but the deficiency of tumor specific antigen, in part, has limited the development of protein vaccine until MAGE antigen was found; Both MAGE-1 and MAGE-3 are widely studied. The expressions of MAGE family proteins deviate in different tumors, and MAGE-1 and MAGE-3 are two of most common tumor antigens, which express in most tumors respectively or jointly but not in normal tissues except the testes and placenta. Moreover, to facilitate APC uptake tumor vaccines and improve the presentation is still needed. In this research, we used HSP70 as the vaccine carrier and MAGE-1 and MAGE-3 as the targeting antigens to construct a fusion protein vaccine, and determine its humoral and cellular immune responses and the antitumor effects.1. The MAGE-1 expressions in tumor tissues, cell lines and stable transfected cells were measured by the method of immunohistochemistry with anti-MAGE-1 serum. The results showed that the expression rates in hepatocellular carcinoma, gastric carcinoma and colon carcinoma were 75%, 57% and 60% respectively. The expressions of MAGE-1 in tumor tissues (n=49) were distinctly higher than the expressions in normal tissues (P<0.01), which was identical with that reported. In addition, the immunohistochemical detection also indicated that the expression of MAGE-1 was higher in discrete carcinomas than in sarcomas (P<0.01). The study demonstrated MAGE-1 expressed in hepatocellular carcinoma cell line (7721, HHCC, HepG2) and renal clear cell carcinoma cell line (786-o), but not astrocytoma cell line (BT-325) and leukemia cell line(HL60). This character was similar to the MAGE-1 expression pattern in tumor tissues, so the same reason may account for it.2. The eukaryotic expression plasmid pIRES2-EGFP-MAGE-l was constructed, and the sequence was identical with that reported in GeneBank. The plasmid was introduced into the mouse melanoma cell B16, and the EGFP was detected by fluorescent microscope and the expression of MAGE-1 was measured by the method of immunohistochemistry. The stable EGFP-MAGE-1 expression B16 cell line was constructed. In this study, we proved that MAGE-1 was expressed in the B16 cells transfected with MAGE-1 (B16-MAGE-1), but not in B16 cells transfected MAGE-3 (B16-MAGE-3).3. HSP70-peptide complex whether isolated from tumor tissues or constructed by gene engineering are mostly likely to contain several tumor special antigenic peptide, and HSP70 was used as rumor vaccine carrier to generate tumor special CTL in vivo, and kill tumor cells. In this study we constructed prokaryotic expression plasmids of pET28a (+) -MAGE-1, pET28a (+) -MAGE-3 and pET28a (+) -HSP70, and no mutations were found after sequencing. MAGE-1, MAGE-3 and HSP70 proteins were expressed in t...
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