B→O血型转变工具酶α-半乳糖苷酶cDNA克隆及表达

Cloning and Expression of Alpha-galactosidase cDNA for Seroconversion from Group B to O

α-半乳糖苷酶是实现 B→O血型转变、制备通用型血的关键工具酶 .利用反转录 PCR方法从中国海南 Catimor咖啡豆中克隆α-半乳糖苷酶 c DNA,插入嗜甲基酵母 P.pastoris分泌表达载体 p PIC9K中 ,转化 P.pastoris GS1 1 5,筛选高表达重组菌株 .经甲醇诱导表达 7d后 ,发酵液总蛋白分泌量约 1 .2 mg/ml,SDS- PAGE呈现约 41 k D特异表达带 ,与专一性底物对 -硝基 -苯基 -α- D-吡喃半乳糖苷反应证明 ,表达产物具有 α-半乳糖苷酶活性 ,最高达到 1 8U/ml.初步实验表明 ,表达的 α-半乳糖苷酶可酶解 B型红细胞 ,成功实现 B→O血型转变 .

Alpha galactosidase is an important enzyme for seroconversion from group B to universal group O of human red blood cells.Its cDNA cloned by RT PCR from green (unroasted) Catimor coffee beans collected in Hainan Province (China),was constructed into the vector pPIC9K designed for secretion expression in P.pastoris. The construct was then transformed into P.pastoris strain GS115. Followed by expression selection,a recombinant strain that exhibited high level production of alpha galactosidase was obtained.After 7 days of continuous methanol induction,it was observed that the total secreted proteins in culture supernatants reached up to 1.2 mg/ml,migrating as the main bands of 41 kD on SDS PAGE.Alpha galactosidase activity was up to 18 U/ml when assayed using p nitrophenyl alpha D galactopyranoside as substrate.The enzyme was then purified and used to treat group B red blood cells for blood conversion.Preliminary evidence suggested that the recombinant alpha galactosidase could convert these cells serologically to group O.