Construction of a human Flt3-ligand gene containing eukaryotic expressing vector and its expression in COS-7 cells

Objective:To construct an eukaryotic expressing vector pIRES1neo/hFL and express hFL in COS 7 cell.Methods:The cDNA encoding soluble human Flt3 ligand(FL) was obtained by RT PCR from the TF 1 cell lines and was inserted into eukaryotic expressing vector pIRES1neo between EcoR I and BamH I sites after sequencing,and then COS 7 cells were transfected with the recombinant by liposome.The transcription and expression of hFL in the transfected COS 7 cells were assayed by RT PCR,ELISA and the experiment of the human umbilical blood CD34 + cell multiplication,respectively,at 72 hours after transfection.Results:It showed that hFL cDNA cloned in our laboratory was 546 bp in length encoding soluble human FL which was in accordance with the report previously.FL gene was transcripted in transfectants,and FL protein with obvious biological activity was highly expressed with 251 ng/(10 6 cell·d) in the supernatant of the transfectants.Conclusion:Human FL in the recombinant vector is proved to be expressed in COS 7 cells and obvious biological activity of hFL in the supernatant of the transfectants was detected.