| Progress in molecular and cellular immunology during the past two decades has advanced our understanding of tumor-host interactions and opened extraordinary opportunities for the development of antitumor immunotherapies. But the lack of tumor specific antigens, the limitation of MHC and the polymorph of tumor cells are the major factors of the low effect of tumor vaccine. Heat shock protein 70 is a conserved protein, which can aid the folding and transport of proteins. Hsp70 plays an important role in the antigen presentation pathway. Some findings suggested Hsp70 could be used as a molecular carrier to enhance the antigenicity of antigen linked to Hsp70. MAGE-1 was the first found tumor specific antigen, which expressed in most tumor cells but not in normal tissues except the testes. In our research, the fusion DNA vaccine and protein vaccine of MAGE-1 and Hsp70 were constructed, and their humoral- and cellular-activating effects were measured, and the antitumor effects of vaccines were evaluated.1. MAGE-1 gene was cloned from the total RNA of HepG2 cells by RT-PCR, and its sequence was identical to the report in GeneBank. The analysis of protein encoded by MAGE-1 gene showed that the first 100aa segment had a high hydrophobicity with a potent helical membranous region. The specificity of MAGE-1 was analyzed by Blast, and the result demonstrated the last lOOaa segment of MAGE-1 had a relatively higher specificity. The full length and segmental expression plasmids of MAGE-1 were constructed and expressed in E coli. The expression product of full length was very low, while the MAGE-1 (97-309aa) could be expressed well. The MAGE-1 (194-309aa) was expressed and purified. The rabbit was immunized with the purified protein, and the antiserum was obtained. The agar gel precipitation of anti-MAGE-1 antiserum showed 1:32 antiserum could form the Ag-Ab precipitation with the GST-MAGE-1 (194-309aa ). After cross-absorbed by GST-Sephrose 4B, the antiserum could bind with the MAGE-1, but not the GST protein. ELISA showed the titer of antiserum was higher than 1:10,000.2. The reverse transcript virus plasmid pLXSN-MAGE-1 was constructed. After virus plasmid was packaged by PA317, the mouse melanoma cell B16 was infected. The transcription of MAGE-1 was detected by RT-PCT, and Western Blot demostrated that a 46kDa protein in the infected cells could be recognized by MAGE-1 antiserum. The Hsp70 gene was cloned from Mycobacterium tuberculosis H37Rv by PCR, and its sequence was identical to that reported except a silente mutation. The eukaryotic expression plasmids of Hsp70, MAGE-1 and their fusion gene were constructed, and the C57BL/6 mice were immunized with DNA vaccines. The results of IFN-gamma ELISPOT andIL-2 ELISA demonstrated that the fusion DNA vaccine could induce a higher number of B16-MAGE-1 specific T cells than other groups (p<0.05). The cytotoxicity determined by LDH release assay showed the spleen lymphocytes separated from the mice immunized with the fusion DNA vaccine had a higher cytotoxicity to B16-MAGE-1, and lower to B16. But the fusion DNA vaccine didn't induce the titers of anti-MAGE-1 antibody (p>0.05). The therapeutic effects to the mice bearing B16-MAGE-1 tumor demonstrated that the fusion DNA vaccine could reduce the growth of B16-MAGE-1 in the mice.3. The Hsp70 and MAGE-1 (97-309aa) were expressed in E coll, and the proteins were purified with GSTrap FF columns. The MAGE-1 and Hsp70 fusion gene prokaryotic expression plasmid (pGEX-MAGE-l-Hsp70) was constructed and expressed in the DH5a. The fusion protein was purified, and the C57BL/6 mice were immunized with Hsp70, MAGE-1 and fusion proteins. The IFN-Y ELISPOT and IL-2 ELISA demonstrated there were relatively more B16-MAGE-1 specific T cells in the fusion protein groups than in other groups (p<0.05). The cytotoxicity measured by LDH release assay showed the spleen lymphocytes from the mice immunized with the fusion protein vaccine had a higher cytotoxicity to B16-MAGE-1. The fusion protein vaccine could induce high...
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