establishment of stable chinese hamster ovary cell line capable of expressing human recombinant hemopexin: a promising therapeutic modality against hemolytic anemia

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.