Construction of plasmid co-expressing FasL and Der p2 gene and its expression in dendritic cells

Objective To construct eukaryotic expression vector co-expressing murine FasL and Der p2 gene,and to detect their expressions in bone marrow-derived dendritic cell(DC).Methods Plasmid plambd-Der p2 as a template,Der p2 gene is amplified using PCR.The plasmid pIRES2EFGP and Der p2 gene were double digested using restriction endonuclease BstX1 and Not1,then Der p2 gene was ligated in pIRES2EGFP(pIRES2EGFP-Der p2).The FasL gene was excised from the plasmid pMD18T-FasL as a BamH1-Sal1 fragment and cloned in pIRES2EGFP-Der p2.Plasmid pIRES2EGFP-FasL-Der p2 containing both FasL gene and Der p2 gene was obtained.After transfected into DC,the expressions of FasL and Der p2 mRNA were detected by RT-PCR,and the expressions of FasL and Der p2 proteins were assayed by Western Blot.Results The cloned full reading frame of FasL and Der p2 cDNA was in coincidence with the sequence registered in GenBank.In the DC after transfection of pIRES2EGFP-FasL-Der p2,the expressions of mRNA and protein of FasL and Der p2 were detected.Conclusion The eukaryotic expression vector pIRES2EGFP-FasL-Der p2 co-expressing FasL and Der p2 gene is constructed successfully,and FasL and Der p2 gene could be expressed correctly in vitro after transfecting DC.