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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。本文报道利用反转录PCR方法从中国海南兴隆咖啡豆中克隆α_半乳糖苷酶cDNA ,插入嗜甲基酵母 pichapastoris表达载体 pPIC9K中 ,转化P .pas torisGS1 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 .2mg/mL ,SDS_PAGE呈现约41kD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_半乳糖苷可酶解B型红细胞 ,成功实现B→O血型转变。本文同时综述了国外有关使用mPEG遮蔽红胞表面抗原、制备通用型血液研究的进展 ,以及我们在此领域的研究工作。
Alpha-galactosidase is a key tool enzyme for B → O blood group transformation and preparation of universal blood. In this paper, α-galactosidase cDNA was cloned from Xinglong coffee bean by reverse transcription PCR and inserted into the pPIC9K expression vector pichia pastoris. The recombinant plasmid was transformed into P. Pas torisGS1 1 5 to screen for the highly expressed recombinant strain. The total protein secretion of the fermentation broth was about 1.2 mg / mL after methanol-induced expression for 7 days, SDS_PAGE showed about 41 kD specific expression band, with the specific substrate of _ nitro-phenyl _α_D_ galactopyranoside reaction Proved that the expression product has α-galactosidase activity, up to 18U / mL. Preliminary experiments show that the expression of α-galactosidase can be enzymatic hydrolysis of B-type erythrocytes, B → O blood group successfully transformed. This article also summarizes the foreign use of mPEG shielding red cell surface antigen, preparation of universal blood research progress, and our research in this area.