Expression And Function Study Of Human PD-Ls-Fc Fusion Proteins

Programmed death ligands(PD-Ls,PD-L1 and PD-L2)play an important role in maintaining immune tolerance.In this study,the fusion genes PD-L1-Fc,PD-L2-Fc,and PD-L1-Fc-PD-L2 containing the extracellular domain of human PD-Ls,hinge region and Fc fragment of human IgGl were expressed through the prokaryotic cells or eukaryotic cells.Preliminary study of the function of these fusion proteins was prepared to provide the basis for further function study and clinical application of PD-Ls-Fc.Methods:PCR was performed to obtain the gene sequences of PD-L1-Fc,PD-L2-Fc and PD-L1-Fc-PD-L2.The PD-Ls-Fc genes were inserted into prokaryotic vector respectively to construct the recombinant plasmids,later,these plasmids were transformed into E.coli Rosetta.The expression of these fusion proteins was induced by IPTG and confirmed by Western Blot.Similarly,the PD-Ls-Fc genes were inserted into lentiviral expression vector respectively.The eukaryotic recombinant plasmid together with its two helper virus plasmids was then co-transfected into the packaged cell 293T.After that,the cell culture supernatant containing virus was harvested and concentrated to infect 293T cells.Cells were selected in 5?g/mL puromycin and cloned by limiting dilution.The expression of PD-Ls-Fc was confirmed by a series of biological techniques such as RT-PCR,Dot-blot and Western Blot.Furthermore,these proteins were purified by affinity chromatography.The ability of PD-Ls-Fc expressed on prokaryotic cells to bind Programmed death-1(PD-1)was determined by Elisa.Cell Counting Kit(CCK)was used to detect the inhibition of PD-Ls-Fc on activated Jurkat T cells.Results:1?Prokaryotic recombinant plasmids pET-27b-PD-L1-Fc,pET-27b-PD-L2-Fc and pET-27b-PD-L1-Fc-PD-L2 were constructed,and the corresponding strains could express target proteins respectively,with approximately 15mg/L of purified proteins.2?Eukaryotic recombinant plasmids pCDH-CMV-MCS-EF1-copGFP-T2A-Puro containing PD-Ls-Fc genes were constructed.By screening,we acquired the 293T/PD-L1-Fc and 293T/PD-L2-Fc cell lines,which stably expressing PD-L1-Fc and PD-L2-Fc respectively,with approximately 0.3mg/L of purified proteins.3?PD-Ls-Fc expressed on prokaryotic cells are bound to PD-1 to some extent as detected by Elisa.4?Cell proliferation results showed that PD-Ls-Fc expressed on prokaryotic cells and eukaryotic cells could inhibit the proliferation of activated Jurkat T cells.However,compared with PD-Ls-Fc expressed on eukaryotic cells,PD-Ls-Fc expressed on prokaryotic cells required higher dosages to inhibit the proliferation of activated Jurkat T cells.Conclusions:Both the prokaryotic expression system and eukaryotic expression system could express PD-Ls-Fc.Moreover,all of the PD-Ls-Fc proteins could inhibit the proliferation of activated Jurkat T cells,which suggested these proteins have biological activity to a certain extent.The results showed that the proteins expressed in this study could be used for further study and clinical.utilization of PD-Ls-Fc.