原核基因工程制备新型重组α-半乳糖苷酶应用于人B→O血型改造的研究  

A Reconstructed B.Fragilis-derived Recombinant α-Galactosidase Developed for Human Blood Type B→O Conversion

作　　者：高红伟[1] 李素波[1] 鲍国强[1] 檀英霞[1] 王玲燕[1] 金泗虎[1] 王颖丽[1] 季守平[1] 宫锋[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《中国实验血液学杂志》2011年第2期503-507,共5页Journal of Experimental Hematology

摘　　要：本研究利用基因工程方法制备脆弱类杆菌来源的新型α-半乳糖苷酶并将其应用于人B→O血型改造。在获得了能够表达细菌α-半乳糖苷酶工程菌株的基础上,从诱导时间、诱导剂浓度两个方面对工程菌株的诱导条件进行优化,获得细菌α-半乳糖苷酶的最佳可溶性表达条件;超声上清经过阳离子交换层析和阴离子交换层析等方法进行纯化。利用纯化后的α-半乳糖苷酶在磷酸盐缓冲液(pH 6.8)中处理B型红细胞2小时,制备O型红细胞。结果表明细菌α-半乳糖苷酶最佳诱导表达条件为:37℃、起始D600为0.8,IPTG浓度为0.1 mmol/L,诱导时间为2小时。纯化后α-半乳糖苷酶的纯度为电泳纯,比活由纯化前的0.42 U/mg提高到了2.1 U/mg。该酶在26℃、接近中性的pH条件(6.8)下经2小时可将B型红细胞改造成O型红细胞,需要的酶量为225μg/ml红细胞。结论:建立了重组细菌α-半乳糖苷酶的表达纯化工艺,制备的重组α-半乳糖苷酶可有效地将B型红细胞改造成O型红细胞,为B→O血型改造提供了更有效的工具酶。This study was aimed to prepare a reconstructed B.Fragilis-derived recombinant α-galactosidase developed for human B to O blood group conversion.Based on the construction of recombinant E.Coli（DE3） which can express α-galactosidase,the inducing time and inducer concentration were optimized for high expression of α-galactosidase.Then,the expression products in supernatant were purified by cation and anion exchange column chromatography.The purified α-galactosidase was used to treat B group red blood cells in phosphate buffer（pH 6.8） for 2 hours to prepare O group red blood cells.The results showed that the optimal inducing conditions for α-galactosidase expression were IPTG 0.1 mmol/L,37℃ and 2 hours.The specific enzyme activity of purified protein increased from 0.42 U/mg to 2.1 U/mg as compared with pre-purification.And,the conditions of B to O blood group conversion were 26℃,pH 6.8（neutral pH condition） and 2 hours.Moreover,225 μg of the enzyme could converse 1 ml B red blood cells to O completely.It is concluded that the technology of expression and purification of recombinant α-galactosidase has been established,and the purified protein can converse B red blood cells to O completely,which means that an effective enzyme conversing B red blood cells to O has been obtained.

关 键 词：基因重组α-半乳糖苷酶 血型改造 B→O血型改造 

分 类 号：R457.11[医药卫生—治疗学] Q789[医药卫生—临床医学]