Establishment of Stable Chinese Hamster Ovary Cell Line Capable of Expressing Human Recombinant Hemopexin: A Promising Therapeutic Modality Against Hemolytic Anemia

Hemolytic anemia is associated with intravascular heme release and oxidative stresses that lead to endothelial dysfunctions. Hemopexin (HPX) is a plasma heme-binding β1-glycoprotein, which plays a pivotal role in heme transfer to hepatocytes and iron recycling. Recently, HPX administration in hemolytic patients attenuated hemolysis-driven oxidative damages and endothelial disorders that led to a rise in the strategy of HPX therapy. Human HPX production using recombinant DNA technology could provide a real alternative to plasma-derived HPX. The purpose of this study was to generate a stable Chinese hamster ovary (CHO) cell line expressing human rHPX.
Total RNA was extracted from HepG2 cells, and HPX gene was amplified by Real Time-Polymerase Chain Reaction (RT-PCR). Then, the HPX gene was cloned in pcDNA3.1 () shuttle vector. The recombinant pcDNA3.1-HPX was transformed to E. coli TOP10 strain. Gene cloning was verified by colony PCR, restriction digestion, and DNA sequencing. The CHO cells were chemically transfected with pcDNA3.1-HPX, and screening was performed by G418 sulfate effective concentration to develop stable single cell clones. The rHPX expression was verified by RT-PCR and Western blot.
Cloning confirmation analyses showed that HPX gene was successfully cloned in pcDNA3.1 () vector. Screening of transfected cells with G418 sulfate enriched the population of single cell clones expressing human rHPX. The RT-PCR and Western blot analyses confirmed rHPX expression in CHO cell line both at transcriptional and translational levels.
Human rHPX protein was stably expressed in CHO cells. This study was a pioneering work for the future production of therapeutic rHPX.