论文部分内容阅读
α-半乳糖苷酶是进行B→0血型改造的工具酶.采用RT—PCR方法,从中国海南Catimor咖啡豆中克隆了全长1.1 kb的α-半乳糖苷酶cDNA,并构建了其表达载体.电穿孔法将载体导入毕赤酵母GS115细胞,筛选出重组α-半乳糖苷酶菌株.离子交换层析纯化出α-半乳糖苷酶,酶比活性从15U/mg提高到28.14 U/mg.进一步鉴定了酶的生物化学性质,其Km=0.275,Vmax=0.014 mmol·L-1·min-1.据此确定了B→0血型改造的条件:pH 5.5～5.6,每毫升红细胞使用100U α-半乳糖苷酶,26℃,4 h.经动物输血实验初步证明,酶解反应后的通用O型血(enzymatically converted group O red blood cells,ECORBC)是安全的.
α-galactosidase is a tool enzyme for B → 0 blood group modification.A total length of 1.1 kb α-galactosidase cDNA was cloned from Catimor coffee bean of Hainan, China by RT-PCR and its expression was constructed The vector was electroporated into the Pichia pastoris GS115 cells and the recombinant α-galactosidase was screened.α-galactosidase was purified by ion exchange chromatography and the specific activity increased from 15 U / mg to 28.14 U / mg. The biochemical properties of the enzyme were further identified as Km = 0.275, Vmax = 0.014 mmol·L-1 · min-1. The conditions for the B → 0 blood group modification were then determined: pH 5.5 to 5.6, 100U α-galactosidase at 26 ℃ for 4 h.The animal blood transfusion experiment initially proved that enzymatically converted group O red blood cells (ECORBC) were safe.