Bicistronic and Stable Expression of Human Coagulation Factor IX and Enhanced Green Fluorescent Protein in Suspension-Adapted Chinese Hamster Ovary Cells

Abstract

Background and Aim: The current treatment for hemophilia B is replacement therapy, which involves the intravenous infusion of human coagulation factor IX (hFIX) purified from plasma or a recombinant form produced in mammalian cells. In this study, using a bicistronic expression system, the stable expression of the hFIX in a serum-free and suspension-adapted Chinese hamster ovary cell line (CHO-s) was investigated. Materials and Methods: A DNA fragment consisting of hFIX, Internal Ribosome Entry Site (IRES) and Enhanced Green Fluorescent Protein (EGFP) nucleotide sequences was cloned into pcDNA3.1 expression plasmid under the control of Cytomegalovirus (CMV) promoter. The bicistronic plasmid was then linearized using BglII restriction enzyme and transfected into CHO-s cells. The transfected cells were treated with geneticin for 14 days. The culture medium of the stable cells was then collected and the expression level of the hFIX were examined using western blotting and ELISA. The coagulation activity was also evaluated by the chromogenic method. Results: The recombinant CHO-s cells resistant to geneticin were observed under a fluorescence microscope in green color, which indicated the expression and accumulation of the EGFP in the cytoplasm of the cells. The results of Western blotting confirmed the expression and secretion of the hFIX into the culture medium. The amount of the secreted hFIX was 150 ng/mL/106cells with a coagulation activity of 5.6 ±0.2 mU/mL. Conclusion: Our findings demonstrated that this bicistronic expression system could simultaneously produce EGFP and hFIX in CHO-s cells. This expression system facilitates selection and isolation of hFIX-expressing cells.

Keywords

• human coagulation factor ix
• chinese hamster ovary cell
• enhanced green fluorescent protein
• bicistronic expression.