【摘要】 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。更多还原

【Abstract】 Objective To construct a eukaryotic coexpression plasmid containing PML-RARα245 gene and hGM-CSF gene using PML-RARα fusion gene segment with the length of 245 bp.MethodsPML-RARα fusion gene segment was amplified from NB4 cell line by RT-PCR and the whole hGM-CSF gene was amplified from pORF-hGM-CSF plasmid by PCR.Both PCR products were cloned into pIRES plasmid,respectively,to construct a recombinant plasmid pIRES-PML-RARα245-hGM-CSF.After being identified by double enzyme cutting and sequence analyzing,the recombinant plasmids were transfected into K562 cells.RT-PCR and dot blot techniques were used to detect the transcription and translation in eukaryotic cells.Results Restriction analysis(Nhe I/Mlu I,Xba I/Sal I) and sequence analysis confirmed that the length and the sequence of the fragments inserted into multi-clone site A and B of pIRES plasmid were absolutely correct.The transcription and translation of these recombinant plasmids in eukaryotic cells could be correctly detected.Conclusion A recombinant eukaryotic coexpression plasmid containing PML-RARα and hGM-CSF genes has been successfully constructed,which will provide more materials for the research of DNA vaccine used in the treatment of acute promyelocytic leukemia.更多还原