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目的构建PML-RARα融合基因与人白介素-2(hIL-2)基因真核双表达质粒。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RT-PCR扩增Jurkat细胞中的hIL-2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,构建真核双表达质粒。利用酶切和序列分析方法验证所构建质粒的正确性。将构建成功的重组质粒转染A549细胞,通过RT-PCR检测重组质粒在真核细胞中的转录情况,利用点杂交、ELISA检测目的蛋白的表达情况。结果NheⅠ/MluⅠ和SalⅠ/NotⅠ双酶切证明重组质粒中含有相应大小的PML-RARα基因片段及hIL-2基因,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。将所构建的pIRES-PML- RARα-hIL-2质粒转染A549细胞后经RT-PCR鉴定表明,插入到重组质粒中的PML- RARα和hIL-2基因能够在真核细胞中进行正常转录。经点杂交和ELISA检测显示重组质粒在真核细胞中可表达hIL-2蛋白。结论成功构建了pIRES-PML-RARα-hIL-2质粒,该重组质粒能够在真核细胞中正常转录并表达PML—RARα和hIL-2蛋白。
Objective To construct the eukaryotic double expression plasmid of PML-RARα fusion gene and human interleukin-2 (hIL-2) gene. Methods The gene fragment of PML-RARα was amplified from the RNA of NB4 cells by RT-PCR. The hIL-2 gene was amplified by RT-PCR and cloned into pIRES Plasmid multiple cloning sites (MCS) A and B, the construction of eukaryotic double expression plasmid. The correctness of the constructed plasmids was verified by restriction enzyme digestion and sequence analysis. The constructed recombinant plasmid was transfected into A549 cells and the transcription of the recombinant plasmid was detected by RT-PCR in eukaryotic cells. The expression of the target protein was detected by dot blot hybridization and ELISA. Results The NheⅠ / MluⅠand SalⅠ / NotⅠ double digestion proved that the recombinant plasmid contained the corresponding gene fragment of PML-RARα and hIL-2 gene. Sequence analysis proved that the inserted fragment in the recombinant plasmid had the correct nucleotide sequence. The constructed pIRES-PML-RARα-hIL-2 plasmids were transfected into A549 cells and identified by RT-PCR. The PML-RARα and hIL-2 genes inserted into the recombinant plasmids could be normally transcribed in eukaryotic cells. Dot blot and ELISA showed that recombinant plasmid could express hIL-2 protein in eukaryotic cells. Conclusion The pIRES-PML-RARα-hIL-2 plasmid was successfully constructed and successfully transfected into eukaryotic cells to express PML-RARα and hIL-2 protein.