Eukaryotic Expression Of Extracellular Domain Of Canine PD-1 And PD-L1 And Screening Of Stable Cell Lines

Programmed death receptor-1(PD-1),one of the immune checkpoint molecules expressed on the surface of T cells,interacting with PD-L1 ligand to mediate the activation of related inhibitory signaling pathway,which plays an important role in immune regulation and immune balance.Under special physiological conditions,such as chronic virus infection or tumor disease,overactivation of the PD-1/PD-L1 signaling pathway can lead to the influence of T cell proliferation,activation inhibition and cytokine secretion,leading to suppression or depletion of T cell immune function.Previous studies have shown that specific antibodies or small molecule blockers targeting PD-1/PD-L1 can effectively restore the inhibition or depletion of T cell immune function,which has gradually become one of the hot targets in the field of tumor therapy for successful clinical application.Dogs are one of the important companion animals of humans and play an indispensable role in human life,and tumor of dog has become one of the life-threatening factors.The PD-1/PD-L1 pathway has been shown to be closely associated with loss of T cell immune function due to canine tumor disease.Blocking the PD-1/PD-L1 pathway may be a new strategy for the treatment of canine tumors.Therefore,focusing on the extracellular domain of the interaction between canine PD-1/PD-L1,the eukaryotic expression system was constructed to screen the stably transfected cell lines to express and purify the eukaryotic expression recombinant proteins of the canine PD-1 and PD-L1 extracellular domains with immunology activity.This study lays a foundation for the study of the preparation and interaction of canine PD-1 and PD-L1 monoclonal antibodies.In this study,we first constructed the vector pc DNA3.1/Myc-His(+)A-Gluc containing the Kozak sequence and Gluc secretion signal peptide.The DNA sequences of the canine PD-1 and PD-L1 extracellular domain proteins were cloned using molecular biology techniques and inserted into the corresponding eukaryotic expression vectors to construct the eukaryotic recombinant expression vector pc DNA3.1/Myc-His(+)A-Gluc-PD-1 and pc DNA3.1/Myc-His(+)A-Gluc-PD-L1.The recombinant eukaryotic expression vectors were transfected into CHO-K1 cells.Western blot was used to verify the protein expression and optimize the transfection conditions.Then,the transient positive cell lines were diluted and cloned by G418 to obtain stable cell lines expressing canine PD-1 and PD-L1 extracellular domain proteins.In addition,the obtained stable cell lines were subcultured for 60 times,and the copy number,growth curve and cell activity of the exogenous genes in the selected cell lines were detected by semi-quantitative PCR and CCK8 technology to further confirm the stable expression of foreign genes in the obtained cell lines.Finally,the stable cell lines were used to express and purify the target proteins,and the purified products were analyzed by Western blot.The results showed that the 432 bp gene encoding extracellular domain protein of canine PD-1 and the 660 bp gene encoding extracellular domain protein of PD-L1 were obtained.The eukaryotic expression vectors of pc DNA3.1/Myc-His(+)A-Gluc-PD-1 and pc DNA3.1/Myc-His(+)A-GlucPD-L1 were successfully constructed.Western blot results showed that the two transfected cell lines successfully expressed canine PD-1 and PD-L1 extracellular domain proteins.Furthermore,the transient transfected cell lines were screened by G418,and the cell lines expressing the extracellular domain proteins CHO-K1/PD-1(CCTCC NO:C202138)and CHO-K1/PD-L1(CCTCC NO:C202139)were stably transfected by limited dilution.The two selected cells still have high target gene copy numbers and good growth activity after 60 passages.The results show that the cell line has good stability and activity.Finally,the culture supernatants of the two selected cells were purified by affinity chromatography.The protein yield of extracellular domain of canine PD-1 is about 7 mg/L,and that of extracellular domain of canine PD-L1 is about2.8 mg/L.Western blot results further showed that the recombinant protein could be recognized by the corresponding commercial antibodies and had good immunoactivity.In this study,we have successfully screened out the cell lines stably expressing the extracellular domain of canine PD-1 and PD-L1,and recombinant proteins of canine PD-1 and PD-L1 with good immune activity were obtained through eukaryotic expression.This will lay a foundation for the preparation of monoclonal antibodies against canine PD-1 and PD-L1 and the study of their interaction mechanism.