Construction of human PD-1-Fc chimeric molecule and its expression and identification in mammalian CHO cell

Objective To construct eukaryotic expression plasmid of human PD-1 extracellular region-hIgG1Fc-pcDNA3.1(+) eukaryotic expression vector, and express the functional fusion protein in mammalian cell. Methods Encoding sequence of human PD-1 extracellular region was amplified, and then inserted together with hIgG1Fc into pcDNA3.1(+) expression vector. The right recombinant was transfected into mammalian CHO cell by lipofectamine reagent. The supernatant of the cultured cell was collected and analyzed by the sandwich ELISA. The fusion protein was purified by HiTrap recombination protein A affinity chromatography. The molecular weight and immune activity of the fusion protein PD-1 were detected by Western blotting and SDS-PAGE. Results The extracellular region of hPD-1 about 727 bp was cloned from human T cell cDNA library, and then inserted together with hIgG1Fc into the eukaryotic expression vector pcDNA3.1(+). After the transfection of recombinant into mammalian CHO cell by lipofectamine reagent, the expression of PD-1 fusion protein was detected in the cultured CHO cell supernatant by the sandwich ELISA. The immune activity of the fusion protein was verified by Western blotting, and its relative molecular weight was about 42 000, which was very close to its expected value. Conclusion The hPD-1-Fc chimeric molecule is constructed and expressed successfully, which provides base for further investigation of the role of PD-1 in immune tolerance and autoimmune disease.