PML-RARα和hGM-CSF双基因表达载体的构建

Construction of the eukaryotic coexpression plasmid containing PML-RARα gene and human GM-CSF gene

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。

Aim： To construct a eukaryotic coexpression plasmid containing PML- RARα gene and human GM -CSF（ hGM -CSF） gene, which was expected to be used as a modified DNA vaccine for acute promyelocytic leukemia. Methods： PML - 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 -RARα - hGM -CSF. The recombinant plasmid were identified by double enzyme cutting and sequence analyzing. Results： Restriction analysis （NheⅠ/MluⅠ, Xba Ⅰ/Sal Ⅰ ） and sequence analysis confirmed that the length and the sequence of the fragments inserted in multi - clone site（MCS） A and MCS B of pIRES plasmid were absolutely correct. Condusion： A recombinant eukaryotic coexpression plasmid containing PML - RARα and hGM -CSF genes was successfully constructed.