ABSTRACT
Hereditary erythrocyte glucosephosphate isomerase (GPI) deficiency is thefourth most common cause of non-spherocytic hemolytic anemia. A case of GPI deficiencywas confirmed after screening a panel of 18 red cell enzymes and GSH. The GPI variantwas characterized by biochemical parameters including GPI activity in erythrocytes andplasma, low substrate activity, electrophoretic mobility, pH optimum, Michaelis constant(Km), thermostability, and substrate analogue (GAL-6-P) utilization rate. A new GPIvariant was found and was designated as GPI-Guangzhou.
ABSTRACT
AIM: To clone human ?-globin gene carrying a thalassemic mutation IVS II654(C→T) and establish a eukaryotic expression system for high-level expression of human ? IVS II654 gene in mouse erythroleukaemia(MEL) cells. METHODS: The fragments of human ? 654 gene isolated from the ? thalassemia patients homozygous for the ? 654 mutation were amplified by PCR, and cloned to plasmid pBGT51. Then, the human ? LCR and ? 654 gene were subcloned from plasmid pBGT51 to the stable mammalian expression vector pcDNA3.1+ together, and the MEL cells were transfected with this vector using commercially available cationic lipid FuGENE6. The MEL cells were induced for further maturation by DMSO and the expression of human ? 654 gene in the MEL cells was identified by RT-PCR. RESULTS: A mammalian expression system of human ? thalassemic mutation ?IVS II654(C→T) was established. CONCLUSION: The level and the reliability of expression of human ? 654 gene in the MEL cells in vitro are similar to that in vivo in human body. This may be a valuable gene therapy model for human ? thalassemic mutation ?IVS II654(C→T).