ABSTRACT
AIM: To investigate human soluble PD-1 (sPD-1) biological activity sPD-1 gene be cloned and expressed in eucaryote cells. METHODS: sPD-1 gene was amplified from human PBMC mRNA by RT-PCR and cloned into eucaryotic expression vector pSG5-Fc. And the positive recombinant plasmid pSG5-Fc -sPD-1 was screened by enzyme digestion and sequencing.The correct sequencing of the recombinant plasmid pSG5-Fc-sPD-1 was transfected into COS-7 cells. The expressed recombinant protein in the supernatant was concentrated with protein A-agarose and analyzed by Western blot. The binding activity to PD-L1 which was expressed with prokaryotic cells was detected with Co-IP. RESULTS: The human sPD-1 fragment was obtained through RT-PCR. The plasmid pSG5-Fc-sPD-1 was constructed by double enzyme digestion and ligation of vector pSG5-Fc and fragment sPD-1. Sequenced sPD-1 gene was coincident with the theoretical sequence. sPD-1-Fc fusion protein in the supernatant was expressed in COS-7 cells and identified by Western blot. The activity of recombinant fusion protein sPD-1-Fc bound to PD-L1 had been detected with Co-IP. CONCLUSION: The human sPD-1 has been cloned and expressed in eucaryote cells successfully. The sPD-1-Fc fusion protein can be effective in binding to PD-L1, which can be used for further research in the function and clinical application of sPD-1.
