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

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.

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

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 exhibited 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.