MAGE-3基因片段真核表达质粒的构建

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

目的克隆人MAGE-3基因片段,以构建真核重组表达质粒pcDNA3.1-MAGE-3,为制备以MAGE-3基因片段为基础的核酸疫苗及探讨相关的肿瘤免疫治疗提供实验依据。方法RT-PCR法从肝癌组织制备出含BamHI、EcoRI酶切位点的MAGE-3基因片段,pGEM-TEasy为克隆载体,pcDNA3.1为真核表达载体,采用DNA重组技术,将目的基因先克隆至pGEM-TEasy载体再亚克隆至pcDNA3.1载体上,然后,据氨苄青酶素(Amp)抗性、蓝白筛选实验、克隆鉴定引物T7/SP6PCR扩增方法鉴定阳性克隆,并对阳性克隆pcDNA3.1-MAGE-3中的插入序列进行DNA测序。结果扩增出了MAGE-3目的基因片段;经蓝白筛选实验、克隆鉴定引物扩增方法鉴定得到重组子pGEM-T-MAGE-3;引物扩增方法鉴定得到重组子pcD-NA3.1-MAGE-3;DNA测序后与GenBank中MAGE-3相应序列比较,结果完全一致。结论成功构建了重组pcDNA3.1-MAGE-3肿瘤核酸疫苗,为肿瘤免疫治疗提供了条件。

[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.