Mammalian expression vector

To generate protein antigen of hPD-1, the ECD of hPD-1 gene was fused in frame with soluble FLAG tag, internal ribosomal entry site (IRES), and enhanced green fluorescence protein (EGFP) which was cloned into a mammalian expression vector (6085–1, Clontech). The construct was transfected into CHO cells and treated with G418 (345,812, EMD Millipore) for 1 week. The EGFP-positive CHO cells were enriched by FACS Aria II cell sorter (BD Biosciences), and the FACS-sorted EGFP-high CHO/hPD-1 ECD cells were cloned by limiting dilution. To select CHO/hPD-1 ECD clones, we measured the levels of EGFP expression and soluble hPD-1 ECD secretion by FACSVerse (BD Biosciences) and anti-FLAG western blot, respectively. The soluble hPD-1 ECD protein was purified from the culture supernatant of CHO/hPD-1 ECD cells by an anti-FLAG M1 agarose affinity gel (A4596, Sigma).
To generate cellular antigen of hPD-1, the gene construct in the pCMV3 mammalian expression vector (Sino Biological Inc.) was transfected into BALB/c mouse-originated CT26 cells by Lipofectamine 2000 reagent (11,668,019, Invitrogen).

Various molecular biology reagents were purchased from Invitrogen Life Technologies (Gaithersburg, MD); Stratagene(La Jolla), CA; and Promega Biotech (Madison, WI). All DNA and protein size markers were purchased from Invitrogen Life Technologies (Gaithersburg, MD). The mammalian expression vector was purchased from Clontech (Palo Alto, CA). Various antibodies were obtained from Cell Signaling Technology (Boston, MA); Roche Molecular Biochemicals (Indianapolis, IN); Santa Cruz Biotechnology(Dallas, TX); Abcam Inc(Cambridge, MA) and Abclonal Inc (Wuhan, China). The culture medium and most other chemicals and antibiotics were purchased from Sigma (St. Louis, MO) and Invitrogen Life Technologies(Gaithersburg, MD).