Eukaryotic expression of human ICOS-Ig fusion protein and analysis of its biological activity / 第二军医大学学报

ABSTRACT

Objective: To express human ICOS extracellular region and IgG Fc fusion protein using euckaryotic vector and to analyze its biological activity. Methods: Human ICOS extracellular region and IgG Fc fragment were cloned by RT-PCR and inserted into eukaryotic expression vector pSecTag2. The constructed plasmid pSecTag2-ICOS-Ig was stably transfected into CHO cells. Soluble ICOS-Ig fusion protein was collected from serum-free medium and purified with protein A affinity chromatography. The purified product was analyzed by SDS-PAGE, Western blotting assay, and ELISA. Fluorescence-activated cell sorting (FACS) and mixed lymphocyte reaction (MLR) were used to study the activity of the fusion protein. Results: ICOS extracellular region and IgG Fc fragment were successfully cloned into expression vector; ICOS-Ig fusion protein was expressed and purified in mammal cells. The purified fusion protein specifically bound to ICOSL and inhibited mixed lymphocyte reaction. Conclusion: A ICOS-Ig fusion protein expression system has been successfully constructed, and bioactive ICOS-Ig fusion protein has been obtained.