Construction eukaryotic expressing plasmid of human melanoma antigen-3 gene fragments

[Objective] To clone human MAGE-3 gene fragments to construct eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-3, to produce nucleic acid vaccine based on MAGE-3 gene fragments, to probe into related tumor immune treatment, and to provid experiment basis. [Methods] MAGE-3 gene fragments containing BamH1,EcoR1 enzyme cutting sites were produced by RT-PCR. Using pGEM-T Easy as clone vector, pcDNA3.1 as eukaryotic expressing vector, and by DNA recombinant technology making the aim gene fragments were cloned into pGEM-T Easy vector and subcloned into pcDNA3.1 vector. Then, according to Ampicillin antibiotic, blute-white screen experiment, clone detective primer T7/SP6 PCR amplifying selected positive clone and sequenced the MAGE-3 aim fragments sequence to be inserted in the middle of the positive clone. [Results] MAGE-3 aim gene fragments were amplified. By blue-white screen experments,clone detective primer T7/SP6 PCR amplifing identified recombinant plasmid pGEM-T-MAGE-3; priner amplifing identified recombinant plasmid pcDNA3.1-MAGE-3. The sequence of MAGE-3 of position recombinant plasmid was compared with the sequence of MAGE-3 of GenBank published after DNA sequenced; and the result was same. [Conclusion] The research successfully constructs recombinant tumor nuleic acid vaccine pcDNA3.1-MAGE-3, and it provides the condition for tumor immune treatment.