黑色素瘤抗原-3基因片段真核表达质粒的构建

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-T Easy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.

Constraction of the eukaryotic expressing plasmid of human melanoma antigen-3 gene fragments

Objective To provide the experimental basis by constructing the eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-3. The study is aimed produce tumor nuclei vaccine and to probe into related immune treatment. Methods By reverse transcripe-polymerase chain reaction (RT-PCR), the aim gene containing BamH1 with EcoR1 enzyme cutting sites of Melanoma antigen-encoding gene-3 (MAGE-3) was produced. Using pGEM-T Easy as the vector, pcDNA3.1 as eukaryotic expression vector, the aim gene were made separately according to the directions for cloning. According to ampicillin (Amp) antibiotic and blut-white screen method, primer T7/SP6 PCR amplification methods was selected for position clone and the aim gene was insert in the middle of the position clone. Results MAGE-3 aim gene was amplified. Recombinant plasmid pGEM-T-MAGE-3 and pcDNA3.1-MAGE-3 were used for identification. The sequence of MAGE-3 of position recombinant plasmid after DNA sequenced was compared with the sequence of MAGE-3 of GenBank published and the result were matched completely. Conclusions The research successfully constructs the recombinant tumor nuclei vaccine pcDNA3.1-MAGE-3 and provides the basis for immune treatment.