Preparation Of Mouse Anti-human PD-1 Monoclonal Antibody And Identification Of Its Biological Characteristic

PD-1 is a co-inhibitory receptor that is a member of the B7 family.It is a 50-55 kDa transmembrane protein and is inducibly expressed on T cells,B cells,NK cells,NKT cells,monocytes and DCs.PD-1 and its ligands,PD-L1 and PD-L2,have an important inhibitory function to play in the regulation of immune homeostasis and in the maintenance of peripheral tolerance.This pathway exerts critical functions in the setting of autoimmune diseases,chronic viral infections and tumors.Various tumours were reported to upregulate PD-L1 expression correlate with poor prognosis.Blockade of this pathway have validated its potential as a new strategy for cancer immunotherapy.We established a gene transfected cell line that expressing the human PD-1 stably and BALB/c mice were immunized with this cell line.We obtained a hybridoma cell line that secreting anti-PD-1 mAb by B lymphocyte hybridoma technique and identified the biological characteristics of this mAb preliminarily.Part 1:Construction of human PD-1 gene transfected cell lineObjective:To construct gene transfected cell line that expressing the human PD-1 stably.Methods:The retroviral vector pEGZ-Term-R and human PD-1 gene were used to construct the recombinant plasmid by restriction endonuclease and T4 DNA ligase.The recombinant plasmid together with its helper virus vector was cotransfected into the 293T cells.The L929 cells were infected with the supernatant of the transfected 293T cells,and then were selected with G418.To obtain gene transfected cell line that expressing the human PD-1 stably by flow cytometry.Results:Gene transfected cell line L929/PD-1 was established.This cell line expressed the human PD-1 stably.Conclusion:Gene transfected cell line that stably expressed the human PD-1 was successfully established.It provided the immunogen for the following experiments and provided a useful tool for the study of PD-1/PD-L1 pathway.Part 2:Preparation of mouse anti-human PD-1 monoclonal antibody and identification of its biological characteristicObjective:To prepare mouse anti-human PD-1 monoclonal antibodies(mAbs)and identify its biological characteristics.Methods:BALB/c mice were immunized with the gene transfected cell line L929/PD-1.The anti-PD-1 mAb was identified with mouse IgG rapid isotyping analysis,recognition profile assay,antibody titer evaluation,competitive inhibition test,western blot and ELISA.Results:A hybridoma cell line that secreting anti-PD-1 mAb was successfully obtained,named 6E1.The heavy chain of mAb was IgG1,and its light chain was κ chain.It could detect PD-1 molecules expressed on U937,Thp-1,Jurkat cell lines and human PBMCs,similar to the commercialization PD-1 mAb(MIH4).The competitive inhibition assay showed that mAb 6E1 bind to the similar epitope with mAb MIH4.Western blot and ELISA results demonstrated that 6E1 could recognize PD-1 protein specificity.Conclusion:Mouse anti-human PD-1 monoclonal antibody was successfully established,which could be used to flow cytometry analysis,western blot and ELISA analysis.It provided a useful tool for the study of PD-1/PD-L1 pathway.