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

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: Human rHPX protein was stably expressed in CHO cells. This study was a pioneering work for the future production of therapeutic rHPX.