The Constructions Of Heat Shock Protein 70 And MAGE-3 Fusion Protein Vaccine And DNA Vaccine And Their Antitumor Effects

Tumors threaten people's health. Tumor vaccines, which can elicit antigen-specific antitumor immunity and play an important role in prevention and therapy of tumor, are regarded as the most attractive method. However, the potency of vaccines is poor owing to the lack of specific antigens and inefficient antigen presentation. The melanoma antigen (MAGE) was the first reported example of tumor specific antigens. MAGE-3 is one important number of MAGE. MAGE-3, expressing in most malignant tumors but not in normal tissue except for testis and placenta, has been used as the ideal target. The MAGE-3 vaccines can induce specific CTL and show potency in tumor immunotherapy. However, there is still a need to enhance the potency of MAGE-3 vaccines. As molecular chaperone, heat shock protein (HSP) participates in processing and presentation of tumor antigen and plays an important role in eliciting antitumor immunity. Recentresearches have demonstrated that linkage of antigens to M. tuberculosis HSP70 leads to the enhancement of tumor vaccines potency.In this research, the HSP70 and MAGE-3 fusion protein and DNA vaccines were constructed and their humoral and cellular antitumor immunity were evaluated. Their therapeutic effects against established tumor expressing MAGE-3 were investigated.1: Cloning, Prokaryotic Expression of Human MAGE-3 Gene and Establishing the Mouse Melanoma Cells Expressing MAGE-3Objective: To amplify human MAGE-3 gene and analyze antigenicity of MAGE-3. To obtain the B16 cell that stably expressing MAGE-3. Methods: The full-length cDNA of MAEG-3 gene was amplified by RT-PCR from human melanoma cell line. Analyze of tumor antigen MAGE-3 using software. Amplified MAGE-3 gene 3'-terminal 360bp segment by PCR. The expression plasmid pET-MAGE-3 was constructed and the BL21(DE3) containing the plasmid was induced. The MAGE-3 gene was cloned into the vector pIRES2-EGFP to construct the pIRES2-EGFP-MAGE-3 plasmid. The plasmid was introduced into the mouse melanoma B16 cells under mediation of lipofectamine. The positive clones were selected by using G418. The expression of EGFP and MAGE-3 mRNA in positive clones were detected by fluorescence microscope and RT-PCR respectively. Results: The full-length cDNA of MAEG-3 gene was amplified by RT-PCR and the sequences were identical with that reported in GeneBank. The C-terminal 120aa segment of MAGE-3 had a high antigenicity. The 3'-terminal 360bp of MAGE-3 was amplified by PCR. The pET-MAGE-3 plasmid was successfullyconstructed and the BL21 (DE3) containing the plasmid could express a 17kD protein whose purity was 85%. The pIRES2-EGFP-MAGE-3 plasmid was constructed and transfected into B16 cell successfully. Green fluorescence of fused protein expression was found and MAGE-3 mRNA was detected in the positive clones. Conclusion: The full-length cDNA of MAEG-3 gene was amplified. The 120aa segment of MAGE-3 encoded by 3'-terminal 360bp was expressed and purified successfully. The B16-MAGE-3 cells that stably expressing MAGE-3 was obtained, which lay a foundation for application of MAGE-3 in tumor immunotherapy.2: The Constructions of HSP 70 and MAGE-3 Fusion Protein Vaccine and Its Antitumor EffectsObjective: To construct HSP 70 and MAGE-3 fusion protein vaccines and investigate its antitumor effects. Methods: The prokaryotic expression plasmids pET-HSP70, pET-HSP70-MAGE-3 were constructed and expressed in E coli. The C57BL/6 mice were immunized with protein vaccines. The spleen lymphocytes secreting IFN-r, the cytotoxicity of CTL to B16-MAGE-3 and anti-MAGE-3 antibody titer in serum were measured by ELISPOT, LDH release assay and ELISA, respectively. The therapeutic effects were investigated. Results: The HSP70 and HSP70-MAGE-3 protein were obtained. The C57BL/6 mice immunized with protein vaccines. There were more frequency of MAGE-3 specific CTL, higher cytotoxicity of CTL to B16-MAGE-3 cells and higher titer of anti-MAGE-3 antibody in serum in the HSP70-MAGE-3 group than those in MAGE-3 group, HSP70 group and MAGE-3+HSP70 g...