Cloning,Gene Expression MAGE-4,MAGE-12 And Distribution Of Tumor Tissues

Objective:MAGE is a member of cancer/testis (CT) antigen family that belongs to tumor specific antigen.The advantages of this family mainly focused on their specificitity,little side effects and convenient operation.Based on the fact that CT expression exist in a wide variety of cancer as well as normal testis and ovaries primarily, it make them became the first candidate for peptide tumor vaccine. Given that the MAGEs(MAGE-4 and MAGE-12) has a wide distribution in different tumor tissues, these molecules are focused on for their protein expression and monoclonal antibody preparation in our experiment.Methods: The coding region of human gene MAGE-4 and MAGE-12 was preparedby RT-PCR and was sub-cloned into the prokaryotic expression vector pGEX-4T-2.After expression in E.coli BL21 cells,the MAGE -4/GST fusion protein was purified by GST affinity chromgraphy.The distribution of MAGE-4, MAGE-12 and some other CT antigens were also investigated by RT-PCR as well as by ELISA in different normal and tumor tissues.The fusion protein was identified by Western Blot.Results: A high level expression of MAGE-4/GST fusion protein with 45 Kdamolecular weight was obtained after IPTG induced in E.coli.The prokaryotic expression vector pGEX-4T-2-MAGE-12 was obtained.Tissues distribution revealed that the MAGE -4 and MAGE -12,which expressed in wide varity of malignant tumor, has little relation with the clinic stage of esophageal cancer.Conclusion: 1 , We prepared the prokaryotic expression vector pGEX-4T-2-MAGE-12 and MAGE -4/GST fusion protein for the preparation of monoclonal antibody and tumor polyvaccine.2, By investigationing their tissues distribution,we described the...