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The construction of eukaryotic vectors that express MUC1/Y and the outcome of modifying dendritic cells to induce specific immunoresponse


, : The construction of eukaryotic vectors that express MUC1/Y and the outcome of modifying dendritic cells to induce specific immunoresponse. Zhonghua Weishengwuxue He Mianyixue Zazhi 24(7): 533-537, July

Objective To develop MUC1 and MUC1/Y gene therapy for breast cancer, we constructed the eukaryotic expression vectors that express a full length MUC1/Y cDNA. To modify DC with these vectors in order to induce specific immunoresponse. Methods To collect the MUC1/Y fill-length cDNA from breast cancer cell line T-47D and then clone it into pIRES2-EGFP expressing green fluorescence protein and eukaryotic expression vector pCDNA3. 1 respectively. To selected 10 breast cancer patients with HLA-A2 phenotype, and then to induce DC by rhIL-4 and GM-CSF in vitro. MUC1/Y-pCDNA3.1 or MUC1-pCDNA3.1 were used to pulse DC, then co-cultured DC with T cell to induce CTL. At the same time, transfected DC with MUC1/Y-pIRES-EGFP to show the transfection efficiency. MCF-7, the breast cancer cell line, which expresses MUC1/Y, MUC1, and HLA-A2, was used as target cell; the cytolytic of specific CTL was measured by LDH-releasing assay. IFN-gamma was quantified by ELISA. Results The results of the sequencing demonstrated that we have cloned MUC1/Y successfully. The transfection efficiency of plasmid was about 10%. The cytolytic activity of T-DC-MUC1-pCDNA3. 1 was about 64%, and the cytolytic activity of T: DC-MUC1/Y-pCDNA3.1 was about 32%. There was a significant difference between these results and those of control group respectively. The results of annexin V-FITC experiments showed that T-DC-MUC1-pCDNA3.1 induced apoptosis of specific target cell. Conclusion Constructed eukaryotic vector pIRES2-EGFP-MUC1/Y that contains full length MUC1/Y cDNA can be used to study the transfection efficiency and select the positive clone; MUC1-pCDNA3.1 and MUC1/Y-pCDNA3.1 can induce powerful CTL immunoresponse, especially MUCl-pCDNA3.1.

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