Expression Of GPx1 In Eukaryotic Cells And Study On Its Antioxidant Effect

Reactive oxygen species(ROS)are produced by cell metabolism in vivo.When the amount of ROS is larger than that of antioxidant defense system,oxidative damage of DNA will appear.The oxidative damage of lipids proteins and nucleotides by ROS may lead to inflammation,cerebral ischemia,mutagenesis,cancer,dementia and physiological aging.Therefore,it is suggested that exogenous antioxidant compounds can be used to improve the occurrence of oxidative damage.Selenium is part of the antioxidant defense system in animals and human and it plays a key role in important metabolic processes.Se has been shown to be part of the glutathione peroxidase activity center.The enzyme protects tissues from oxidative stress by catalyzing peroxides caused by various of cell damage.Glutathione peroxidase 1(GPx1)is widely present in all tissues and is thought to play a role in antioxidant defense.Although the prokaryotic expression of glutathione peroxidase is mature,the prokaryotic expression of glutathione peroxidase is prone to mismatch and is not accurate enough.Eukaryotic cells have a complex regulatory system of post-translational protein modification,which can accurately express the correct spatial conformation of proteins.The biological activity of eukaryotic proteins expressed by eukaryotic system is very close to that of natural proteins.Therefore,the preparation of glutathione peroxidase,which can be expressed in eukaryotic,has a wide application prospect,and it provides a basis for future eukaryotic expression of selenoprotein.In this study,eukaryotic expression of GPx1 was carried out by eukaryotic incorporation mechanism of selenocysteine.pSelExperss1 was selected as expression vector.Hek293 T cells were widely used in transient transfection because of their easy culture and high transfection efficiency.At the same time,it has a complete biochemical mechanism of post-translational modification and folding,and it can express foreign proteins efficiently,and it also has some advantages in the expressionof selenoprotein.The eukaryotic expression of selenium protein was identified by Western blot.The antioxidant properties of GPx1 were also studied by using MDA kit and LDH kit.Through experiments,A relatively clear special band appeared after Western blot experiment,which proves that GPx1 can successfully express selenium-containing protein in Hek293 T cells.It is not difficult to find that the value of GPx1 prevention group and GPx1 treatment group is better than that of the experimental control group treated with hydrogen oxide alone in the study of antioxidant activity.This means that GPx1 does have a good effect on the antioxidant activity of cells.In conclusion,GPx1 was expressed in eukaryotic cells successfully in this study,and it is confirmed that GPx1 has antioxidation effect on cells,which furtherly indicates the necessity of eukaryotic expression of GPx1.This will play an important role in promoting its clinical application as a drug in the future.