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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.
The removal of B antigen from erythrocytes by α-galactosidase is an effective method to prepare universal O-type erythrocytes.In this paper, the physical and chemical properties of purified O-glycans from Bacteroides fragilis were studied by cloning and expressing, The molecular weight of the enzyme is 64908Da, the isoelectric point is between 7.12 and 7.30, the optimal temperature is 41 ℃ and the optimum pH is 5.6-6.0. Its physicochemical properties are suitable for the blood group transformation of type B erythrocytes. In order to determine the efficiency and rapidity , Mild hydrolysis conditions, the paper optimized the process of enzymatic hydrolysis of B-type erythrocytes.By studying the impact of buffer on the binding of enzyme and erythrocytes, we determined that the optimal enzymolysis buffer was 250mmol / L glycine and 3mmol / LNaCl, pH6.8, the optimal hematocrit of enzymolysis was 40%, the temperature of enzymolysis was 26 ℃, the enzymolysis time was 1h.The morphological and functional indexes of B-ECORBCs obtained by the optimized enzymolysis process were normal, The result of cell proved that the labeling rate of B antigen and H antigen was comparable with that of O-type erythrocytes, which indicated that the process of preparing B-ECORBCs was mature.The method has the advantages of less enzyme dosage, mild enzymatic hydrolysis conditions, simple preparation process and short time, Has a good clinical application King.