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目的:构建急性早幼粒细胞白血病(APL)PML-RARα基因与人粒巨噬细胞集落刺激因子(hGM-CSF)基因真核双表达载体,为利用DNA疫苗治疗APL奠定基础。方法:利用RT-PCR技术从NB4细胞的RNA中扩增出PML-RARα融合点附近的部分基因片段,通过PCR从pORF-hGM-CSF质粒中扩增出hGM-CSF基因,并分别将两基因片段连接到pIRES质粒的多克隆位点(MCS)A和B中,构建真核双表达载体。利用酶切和序列分析方法验证所构建载体的正确性。结果:NheI/M luI和XbaI/SalI双酶切证明重组质粒中含有相应大小的PML-RARα基因片段及hGM-CSF基因,且序列分析证明重组质粒中插入片段的碱基序列均完全正确。结论:成功构建了含有PML-RARα基因及hGM-CSF基因的真核双表达载体。
Objective: To construct eukaryotic double expression vector of PML-RARα gene and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene in acute promyelocytic leukemia (APL), and to lay the foundation for the treatment of APL with DNA vaccine. Methods: The partial gene fragment near the fusion site of PML-RARα was amplified from the RNA of NB4 cells by RT-PCR. The hGM-CSF gene was amplified by PCR from pORF-hGM-CSF plasmid and the two genes The fragment was ligated into the multiple cloning site (MCS) A and B of the pIRES plasmid to construct a eukaryotic double expression vector. The correctness of the constructed vector was verified by digestion and sequence analysis. Results: NheI / MluI and XbaI / SalI double digestion showed that the recombinant plasmid contained the corresponding gene fragment of PML-RARα and hGM-CSF, and the nucleotide sequence of the inserted fragment in the recombinant plasmid was completely correct. Conclusion: The eukaryotic double-expression vector containing PML-RARα gene and hGM-CSF gene has been successfully constructed.