AIM: To construct an eukaryotic superantigen gene expression vector containing the recombinant gene of SEA and CD80 molecule transmembrane region (CD80TM), and to expresss taphylococcus enterotoxin A (SEA) on the memb...AIM: To construct an eukaryotic superantigen gene expression vector containing the recombinant gene of SEA and CD80 molecule transmembrane region (CD80TM), and to expresss taphylococcus enterotoxin A (SEA) on the membrane of hepatocellular carcinoma (HCC) cell to form a superantigen gene modified tumor vaccine for HCC. METHODS: SEA and linker-CD80TM gene were amplified through PCR from plasmid containing cDNA of SEA and CD80.Gene fragments were then subcloned into the multiple cloning sites of retroviral vector pLXSN. Recombinant plasmid was transferred into HepG2 cells mediated with lipofectamine,positive clones were selected in culture medium containing G418. RT-PCR and indirect immunofluorescence studies confirmed that SEA was expressed specifically on HCC cell membrane. INFT-ELISPOT study demonstrated that SEA protein was expressed on the membrane of HCC cells.Cytotoxicity of HepG2-SEA primed CTLs (SEA-T) was analyzed by^51Cr release assay. T cells cultured with rhIL-2 (IL-2-T) were used as control. RESULTS: Restriction digestion and sequence analyses confirmed the correctness of length, position and orientation of inserted fusion genes. SEA was expressed on the surface of HepG2 cells, HepG2-SEA had strong stimulating effect on production of HepG2 specific CTL (P<0.001). SEA-T had enhanced cytotoxicity to HepG2 cells (P<0.05). CONCLUSION: Tumor cell membrane expressed superarfdgen can be used to reinforce the immune effect of tumor cell vaccine for HCC, which provides a new method of the enhanced active immunotherapy for HCC.展开更多
基金Supported by National Natural Science Foundation of China,No.30271474 and No.39770827
文摘AIM: To construct an eukaryotic superantigen gene expression vector containing the recombinant gene of SEA and CD80 molecule transmembrane region (CD80TM), and to expresss taphylococcus enterotoxin A (SEA) on the membrane of hepatocellular carcinoma (HCC) cell to form a superantigen gene modified tumor vaccine for HCC. METHODS: SEA and linker-CD80TM gene were amplified through PCR from plasmid containing cDNA of SEA and CD80.Gene fragments were then subcloned into the multiple cloning sites of retroviral vector pLXSN. Recombinant plasmid was transferred into HepG2 cells mediated with lipofectamine,positive clones were selected in culture medium containing G418. RT-PCR and indirect immunofluorescence studies confirmed that SEA was expressed specifically on HCC cell membrane. INFT-ELISPOT study demonstrated that SEA protein was expressed on the membrane of HCC cells.Cytotoxicity of HepG2-SEA primed CTLs (SEA-T) was analyzed by^51Cr release assay. T cells cultured with rhIL-2 (IL-2-T) were used as control. RESULTS: Restriction digestion and sequence analyses confirmed the correctness of length, position and orientation of inserted fusion genes. SEA was expressed on the surface of HepG2 cells, HepG2-SEA had strong stimulating effect on production of HepG2 specific CTL (P<0.001). SEA-T had enhanced cytotoxicity to HepG2 cells (P<0.05). CONCLUSION: Tumor cell membrane expressed superarfdgen can be used to reinforce the immune effect of tumor cell vaccine for HCC, which provides a new method of the enhanced active immunotherapy for HCC.
