Study on Construction and Expression of the Eukaryotic Coexpression Plasmid Containing PML-RARα 245-hIL-2

Objective:To construct a eukaryotic coexpression plasmid containing PML-RARα gene by using PML-RARα gene segments with different length.Methods:PML-RARα fusion gene segments with different length were amplified from NB4 cell line by reverse transcription polymerase chain reaction(RT-PCR).Both PCR products were cloned into pIRES plasmid to construct a recombinant plasmid pIRES-PML-RARα.The recombinant plasmids were identified by double enzyme cutting and sequence analyzing.After being transfected into K562 cells,RT-PCR and real-time fluorescence quantitative PCR were used to detect the transcription of these recombinant plasmids in eukaryotic cells.Results:Restriction analysis(Nhe Ⅰ/Mlu Ⅰ) and sequence analysis confirmed that the length and the sequence of the fragments inserted in multi-clone site(MCS) A of pIRES plasmid were absolutely correct.Both mRNA expressed by recombinant plasmids in K562 cell lines could be correctly detected.Conclusions:Recombinant eukaryotic coexpression plasmid containing different PML-RARα segments were successfully constructed.And these recombinant plasmids have normal function of transcription in eukaryotic cells in vitro,which will provide more rich materials for the research of DNA vaccine used in the treatment of acute promyelocytic leukemia(APL).