【摘要】 目的构建黑色素瘤抗原-3(M AGE-3)目的基因真核重组表达载体pcDNA 3.1-M AGE-3。方法用RT-PCR法从肝癌组织制备含B amHⅠ、E coRⅠ酶切位点的M AGE-3目的基因,采用DNA重组技术将目的基因克隆至pGEM-T E asy载体和亚克隆至pcDNA 3.1载体上,根据氨苄青霉素(Am p)抗性、蓝白筛选实验、引物扩增等方法筛选、鉴定阳性克隆,并对其中的插入序列M AGE-3目的基因进行DNA测序。结果扩增出M AGE-3目的基因;重组质粒pcDNA 3.1-M AGE-3中的插入片段经DNA测序后与G enB ank中M AGE-3相应序列比较,结果完全一致。结论成功构建了真核重组表达载体pcDNA 3.1-M AGE-3,为肿瘤免疫治疗提供了条件。更多还原

【Abstract】 Objective: To construct MAGE-3 aim gene eukaryotic recombinant expression plasmid pcDNA(3.1)-MAGE-3.Methods: MAGE-3 aim gene containing BamHⅠ,EcoRⅠ enzyme cutting sites was produced from liver cancer tissues by RT-PCR.The aim gene was orientating cloned into pGEM-T Easy vector and subcloned into pcDNA3.1 vector by DNA reconbinant technical.Ampicillin(Amp) antibiotic,blue-white screenexperiment,primer amplify methods were used to selected and identified positiveclone,and the sequence of MAGE-3 aim fragments to be inserted in the middle ofthe position clone was sequenced.Results: MAGE-3 aim gene fragments were amplified.The sequence of MAGE-3 aim gene in the middle of positive recombinant plasmid was compared with the relevant sequence of MAGE-3 of GenBank published after DNA sequenced;and the result was the same completly.Conclusion: The researchsuccessfully constructs eukaryotic recombinant expression plasmid pcDNA3.1-MAGE-3,and this provides the condition for tumor immune treatment.更多还原