Study On Construction And Expression Of The Eukaryotic Coexpression Plasmid Containing PML-RARα Gene And Human GM-CSF/IL-2 Gene

Objective: To construct a eukaryotic coexpression plasmid containing PML-RARαgeneand human GM-CSF/IL-2 gene and detect the protein expressed by these coexpressionplasmids in eukaryotic cells so as to lay the foundation for the research of DNA vaccine forAPL.Methods: PML-RARαfusion gene segments were amplified from NB4 cell line by RT-PCRand were inserted into the MCS A of pIRES plasmid to construct a recombinant plasmidpIRES-PML- RARα. The whole hGM-CSF gene and hIL-2 gene were amplified frompORF-hGM-CSF plasmid or Jurkat cell line by PCR or RT-PCR. Both PCR products wereinserted into the MCS B of pIRES-PML- RARαplasmid respectively to construct two kinds ofrecombinant plasmids pIRES-PML- RARα- hGM-CSF and pIRES-PML-RARα-hIL-2. Allrecombinant plasmids were identified by double enzyme cutting and sequence analyzing beforebeing transfected into K562 or A549 cell lines. The information about transcription ofrecombinant plasmids in eukaryotic cells were detected by RT-PCR and Realtime-PCR. Theprotein translated from recombinant plasmids in eukaryotic cells were detected by ELISA,dotblotting and SDS-PAGE respectively.Results: The results of double enzyme cutting and sequence analysis confirmed thatpIRES-PML -RARα,pIRES-PML-RARα-hGM-CSF and pIRES-PML -RARα-hIL-2recombinant plasmids were successfully constructed. And the length and sequence of thefragments inserted in multi-clone site(MCS) A or MCS B of all the recombinant plasmids wereabsolutely correct. Both mRNA and protein expressed by recombinant plasmids in K562 orA549 cell lines could be correctlly detected.Conclusion: pIRES-PML-RARα,pIRES-PML-RARα-hGM-CSF and pIRES-PML-RARα-hIL-2 recombinant plasmids were successfully constructed, And these plasmids havenormal function of transcription and translation in eukaryotic cells in vitro.