Study On Immunity Of Recombinant Multiple Epitopes Antigen Of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most common malignant tumor which severely harm to human health. Therefore, it's of great importance to set about research of prevention and therapy on HCC in china. In order to pave the way for developing immunotherapy of HCC, we choose T-cell epitopes from AFP and MAGE-1 to produce the recombinant poly-epitope protein antigen and recombinant plasmid DNA and then research their immunity.Method: (1)The gene of multi-epitope were gained with fitly design and it was cloned into prokaryotic expression vector and expressed in E.coli. (2)An recombinant eukaryotic expression vector was constructed and its eukaryotic expression was determined in trensfected cells. (3)The human AFP and MAGE-1 genes were amplified by RT-PCR and the stably expressing transgenic SP2/0 cell lines were established. (4)Balb/c mice were immunized with different forms of antigen. The antibody titer was tested by ELISA. ELISPOT and LDH-release assay showed its T-cell response. (5)Balb/c mice were subcutaneous injected with SP2/0 cells and the protective activity for the pre-immunized mice was observed.Results: (l)The recombinant prokaryotic plasmid was correctly constructed. The gene was expressed in E. coli. highly and the expressed protein was purified efficiently. The recombinant protein Tat+T can can be effectively transduced into mammalian cells(SP2/0 and BHK cell) in vitro. (2)The recombinant eukaryotic expression plasmid was correctly constructed and can expressed in SP2/0 cell. (3)The transgenic SP2/0 cell lines stably expressing of human AFP and MAGE-1 were established successfully. (4)Balb/c mice were immunized with the purified protein and plasmid DNA. The high titer antibody was induced, especially in mice immunized with recombinant protein T. (5)The antigen specific T cell response such as IFN-γ release and cytotoxicity to target cells was much stronger in protein Tat+T group or plasmid prime-protein boost group than in the other groups. (6)In immunized mice, the protective efficiency against transgenic SP2/0-M and SP2/0-A cells, such as inhibition of tumor growth and...