The Study Of Animal Transfusion Experiment On Bâ†'O Blood Conversion

a-galactosidase(EC3.2.1.22)is an exoglycosidase distributed widely in nature and is responsible for cleavage of a-linked terminal galactose residues from glycoconjugates. Our group had cloned the 1.1 kb cDNA of a-galactosidase from Hainan Catimor coffee bean by RT-PCR method. We have constructed a vector for a-galactosidase cDNA expression and transferred a-galactosidase cDNA into Pichia Pastoris GS115 cells by electroporation. Positive clones were selected to secret recombinant a-galactosidase into the culture medium. The recombinant enzyme was purified by cation exchange chromatography and the activity was up to 28.14U/mg. The Km was 0.275 mmol/L, Vmax was 0.014 mmol/L /min.Fresh type B human erythrocyte were washed with PBS buffer (pH=5.5-5.6) and 100U/ml of recombinant enzyme was added into the erythrocyte suspensions, incubated at 26C for 2 hrs. Hemagglutinins assay showed that the B antigens were removed from B erythrocytes efficiently.In order to perform the animal transfusion experiment before entering clinical trial of B→O Blood Conversion, Rhesus monkey and gibbon red blood cells were digested by recombinant a-galactosidase. The results showed that a-galactosidase could remove like-human-Bantigen located on rhesus monkey red blood cell surface as well as B antigen located on the surface of gibbon red blood cells efficiently. There were no harmful effects on donor blood cellular structure and function as well as on donor cell survival in recipient like A rhesus monkey and A gibbon after a-galactosidase digestion. The results also demonstrated that no transfusion rejection happened in recipient rhesus monkey and gibbon with different blood group. The routine blood and urine tests of recipient rhesus monkey and gibbon were normal after enzymatically converted blood cell transfusion. The animal experiment showed that transfusion of enzymetically converted group O Red Blood cells (ECORBC) were perfectly safe.