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目的构建真核重组表达质粒pcDNA-3.1-MAGE-3,为制备肿瘤核酸疫苗及探讨相关的免疫治疗提供实验依据。方法采用逆转录!聚合酶链反应(RT-PCR)法从肝癌组织中制备出含BamHI、EcoRI酶切位点的黑色素瘤抗原-3(MAGE-3)目的基因;pGEM!TEasy为克隆载体,pcDNA3.1为真核表达载体,将目的基因分别先后定向克隆至其上;根据氨苄青霉素(Amp)抗性、蓝白筛选实验、引物T7/SP6PCR扩增方法鉴定阳性克隆,并对其中的插入序列进行DNA测序。结果成功扩增出目的基因;经鉴定得到重组质粒pGEM-T-MAGE-3和pcDNA3.1-MAGE-3;DNA测序后与GenBank中MAGE!3相应序列比较完全一致。结论成功构建pcDNA3.1!MAGE!3肿瘤核酸疫苗,可为免疫治疗提供实验依据。
Objective To construct eukaryotic recombinant plasmid pcDNA-3.1-MAGE-3 and provide experimental evidence for preparation of tumor nucleic acid vaccine and related immunotherapy. Methods The target gene of melanoma antigen-3 (MAGE-3) containing BamHI and EcoRI restriction sites was prepared by reverse transcription polymerase chain reaction (RT-PCR) from hepatocellular carcinoma tissue. PGEM! TEasy was a cloning vector, pcDNA3.1 was used as the eukaryotic expression vector and the target gene was cloned into it respectively. According to ampicillin (Amp) resistance, blue-white screening experiment and primer T7 / SP6 PCR amplification method, the positive clones were identified, Sequence DNA sequencing. The recombinant plasmid pGEM-T-MAGE-3 and pcDNA3.1-MAGE-3 were successfully identified. The sequence of MAGE! 3 in GenBank was the same after DNA sequencing. Conclusion The successful construction of pcDNA3.1! MAGE! 3 tumor nucleic acid vaccine can provide experimental evidence for immunotherapy.