一种来源于脆弱类杆菌的α-半乳糖苷酶的理化性质及其酶解B型红细胞的工艺研究  

Properties of a Novel α-galactosidase from B.Fragilis and Its Potential for Human Blood-type B to O Conversion

作　　者：高红伟[1] 李素波[1] 鲍国强[1] 檀英霞[1] 王颖丽[1] 章扬培[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：《中国科学：生命科学》2011年第10期1030-1036,共7页Scientia Sinica(Vitae)

基　　金：国家自然科学基金(批准号:30671538)资助项目

摘　　要：利用α-半乳糖苷酶去除红细胞表面的B抗原是制备通用O型红细胞的有效方法.本文在克隆表达纯化脆弱类杆菌来源的α-半乳糖苷酶的基础上对其理化性质进行了研究,该酶的分子量为64908Da,等电点在7.12～7.30之间,最适温度为41℃,最适pH为5.6～6.0,其理化性质适合用于B型红细胞的血型改造;为了确定高效、快速、温和的酶解条件,本文对酶解B型红细胞的工艺进行了优化.通过研究缓冲液对酶与红细胞结合的影响,确定了最佳酶解缓冲液为250mmol/L甘氨酸和3mmol/LNaCl,pH6.8;酶解的最适红细胞压积为40%,酶解温度为26℃,酶解时间为1h.利用优化的酶解工艺获得的B-ECORBCs形态及结构功能指标均正常,流式细胞结果证明其B抗原和H抗原标记率与O型红细胞相当,说明制备B-ECORBCs的工艺已成熟.这种工艺具有酶用量少、酶解条件温和、制备过程简单和时间短等优势,具有很好的临床应用前景.Enzymatic removal of blood group B antigen is an effective method to develop universal red blood cells（RBCs） by α-galactosidase.Here we investigated the physicochemical properties of a novel α-galactosidase from B.Fragilis and the optimization of enzymatic conversion for RBC of blood group B to O.The results showed that α-galactosidase hexhibited maximum activity at a broad optimal pH of 5.6-6.0 and an optimum temperature of 41°C.Furthermore,the enzyme was efficient in complete removal of B antigen.The conditions for B to O blood group conversion were 26°C,pH 6.8（250 mmol/L glycine and 3 mmol/L NaCl） for 1 h.The structure and function indexes of the converted red blood cells showed no significant difference from those of normal RBCs.Consequently,the novel α-galactosidase described here was more suitable for enzymatic conversion for achieving the goal of producing universal RBCs,which would improve the blood supply while enhancing the safety of clinical transfusions.

关 键 词：基因重组α- 半乳糖苷酶 脆弱类杆菌 理化性质 血型改造 

分 类 号：R341[医药卫生—基础医学]