1.Department of Gastroenterological Surgery, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325015; 2.Instiute of Molecular Virology and Immunology, Department of Medical Microbiology and Immunology, Wenzhou Medical University, Wenzhou, 325035
LYU Kaiji,CHEN Xudong,CHENG Kai, et al. Prokaryotic expression of murine PD-1 and its ligand PD-L1 extracellular domain gene and their affinity[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2018, 48(6): 401-407，412.
Abstract：Objective: To prepare mouse PD-1 (mPD-1) and its ligand PD-L1 (mPD-L1) protein by prokaryotic expression system and mPD-L1 rabbit polyclonal antibody to verify in vitro the binding affinity of mPD-L1 and mPD-1. Methods: The recombinant plasmids pET21a/mPD-L1 and pGEX 4T-1/mPD-1 were constructed respectively by molecular cloning. Then the recombinant protein of PD-L1 and PD-1 was purified by Ni-NTA affinity chromatography and GST Agarose beads before it was confirmed by SDS-PAGE and Western blot. The purified mPD-L1 extracellular domain protein was immunized with healthy Japanese white rabbits to prepare rabbit polyclonal serum antibody. The titer and specificity of the obtained polyclonal antibody were analyzed by ELISA and Western blot, and the polyclonal antibody was used as positive control in immunofluorescence. Results: SDS-PAGE and Western blot results showed the expected size of the prokaryotic expression of the recombinant protein and mPD-L1 and mPD-1 were 25 kDa and 40 kDa respectively Japanese white rabbits immunized with mPD-L1 protein could produce specific antibodies with its titer peaked at 6 weeks after immunization. Western blot also showed that polyclonal serum could specifically recognize mPD-1. ELISA results showed that prokaryotic mPD-L1 protein could bind mPD-1 protein, with the experimental group OD450 reading significantly higher than the control group. Immunofluorescence results showed that green fluorescent mass seen in the membrane area of B16 cell (mouse melanoma cell) line which expressed PD-L1. Conclusion: mPD-L1 and mPD-1 proteins obtained after prokaryotic expression and then purified, and successfully verified the binding characteristics between mPD-L1 and mPD-1 in vitro, which provided the basis for the later study of the biological characteristics of mPD-1 and PD-L1.
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