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目的采用长度为245bp的急性早幼粒细胞白血病PML-RARα融合基因片段构建新的PML-RARα与hGM-CSF真核双表达载体。方法用RT-PCR技术从NB4细胞的RNA中扩增长度为245bp的PML-RARα基因片段,PCR技术从pORF-hGM-CSF质粒中扩增出hGM-CSF基因,分别将两基因片段连接到pIRES质粒的多克隆位点A和B中,构建真核双表达载体。用酶切和序列分析方法验证所构建载体的正确性。将重组质粒转染K562细胞,利用RT-PCR和点杂交技术检测重组质粒在真核细胞中的转录和翻译情况。结果双酶切结果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,序列分析证明重组质粒中插入片段的碱基序列完全正确。该重组质粒能够在真核细胞中正常转录和翻译。结论成功构建了含有PML-RARα245及hGM-CSF的双表达载体,为筛选合适的抗原片段用于构建治疗急性早幼粒细胞白血病的PML-RARαDNA疫苗提供重要资料。
Objective To construct a new eukaryotic expression vector for PML-RARα and hGM-CSF by using a 245 bp PML-RARα fusion gene fragment. Methods The 245 bp PML-RARα gene fragment was amplified from RNA of NB4 cells by RT-PCR. The hGM-CSF gene was amplified by PCR from pORF-hGM-CSF plasmid and ligated into pIRES Plasmid multiple cloning sites A and B, the construction of eukaryotic double expression vector. The correctness of the constructed vector was verified by restriction enzyme and sequence analysis. The recombinant plasmids were transfected into K562 cells, and the transcription and translation of the recombinant plasmids in eukaryotic cells were detected by RT-PCR and dot blot hybridization. Results The results of double enzyme digestion showed that the recombinant plasmid contained the corresponding fragments of PML-RARα gene and hGM-CSF gene. Sequence analysis showed that the inserted fragment was completely correct in the recombinant plasmid. The recombinant plasmid is capable of normal transcription and translation in eukaryotic cells. Conclusion The double expression vector containing PML-RARα245 and hGM-CSF was successfully constructed and provided important information for screening the suitable antigen fragment for constructing PML-RARα DNA vaccine for acute promyelocytic leukemia.