Construction, eukaryotic expression, and characterization of human OX40-Fc molecule

Objective To construct eukaryotic expression plasmid of hOX40-Fc gene and stably express hOX40-Fc fusion protein with biological activity. Methods The extramembrane encoding region of OX40 molecule was cloned from a normal human activated T cells cDNA library by polymerase chain reaction. After sequencing, the extramembrane encoding region together with human IgG1-Fc cDNA was inserted into the eukaryotic expression plasmid pcDNA3. The right recombinant was transfected into COS-7 cells with lipofectamine reagent. OX40-Fc protein was purified by recombinated protein A affinity column chromatography. The molecular weight, purity, and antigenicity of OX40-Fc were identified by sandwich ELISA, SDS-PAGE, and Western blotting. Results The open-reading frame of OX40-Fc gene was coincident with what we had expected. OX40-Fc protein expression in COS-7 cells was confirmed, and the antigenicity of the purified OX40-Fc protein was analyzed. The purified OX40-Fc protein could inhibit the growth of activated Jurkat cells by proliferation assay. Conclusion The vector is constructed successfully and a purified recombinated OX40-Fc protein is obtained. This lays a foundation for further studies of OX40, such as its role in autoimmune diseases and immune homeostasis.