Expression And Purification Of MIL-21-hIgGFc Fusion Protein In293E Ccells And Its Effects On On CD8~+T Cell Phenotype

Objective To express recombinant protein mIL-21-hIgGFc in293Ecells，and investigate its effect on CD8+T cell.Methods Total RNA was extracted from the mouse spleen cells，and then IL-21gene was amplified by RT-PCR and inserted intoexpression vector PTT3-hIgGFc. We transfected PTT3-mIL-21-hIgGFcinto293E cells by calcium phosphate method. The supernatants werecollected at48hours and72hours and concentrated by MOLLIPORE TFFLabscale TM system (5kd membrane). The mIL-21-hIgGFc fusion proteinwas purified with HiTrapTM Protein G column. The protein was quantifiedby SDS-PAGE and ELISA. The biological activity of the protein wasdetermined by detecting the change of the phenotypes of CD8+T cellstreated with the protein. Results The constructed recombinant plasmidPTT3-mIL-21-Fc was confirmed by sequencing. PTT3-mIL-21-Fc wastransfected into293E cells, mIL-21-Fc protein in culture supernatant wascollected after48hours and72hours. The protein in cell supernatantreached a concentration of787ng/ml which was determined by Elisa. Theprotein was purified by Protein G chromatography column. We treated P1A–specific T cells with mIL-21-hIgGFc, and found that the CD44lowCD62LhiCD8+population increased compared to the control. Conclusion We built PTT3-mIL21-hFc recombinant plasmid,expressed mIL21-hFc fusion protein in293E cells, and purified by ProteinG column. By treating mIL-21-hFc,the antigen-primed CD8+T cells preferto differentiate into CD44lowCD62LhiCD8+T cells which had beenreported as a memory stem phenotype. This protein may be used toimprove the effectness of adoptive T cell cancer therapy.