基因工程人丁酰胆碱酯酶的纯化及其在体外对琥珀酰胆碱的解毒作用(英文)

目的　开发丰富又安全的人丁酰胆碱酯酶来源 ,并检测其解毒效率。方法　家蚕血淋巴液中重组人丁酰胆碱酯酶 (rhBChE)的纯化采用批量硫酸铵分段分离和普鲁卡因酰胺 脂糖凝胶 4B亲和层析技术。结果　聚丙烯酰胺凝胶电泳纯的rhBChE从血淋巴液中的总收率为 6 6 % ,酶比活性达 715mmol·min- 1·g- 1蛋白。rhBChE在 - 2 0℃含 2 0 %甘油及0 .0 2 %NaN3 的 10mmol·L- 1磷酸盐缓冲液 (pH 7.4 )中存放 1年 ,酶活性无明显变化。 18只小鼠ip事先与rhBChE孵温的 1.5LD的琥珀酰胆碱 ,不出现任何中毒症状和体征 ,全部存活 ;而琥珀酰胆碱对照组 ,18只小鼠均剧烈抽搐并在 2～ 5min内窒息死亡。结论　实验结果说明 ,从感染家蚕幼虫血淋巴液中纯化rhBChE的方法可靠 ,基因工程rhBChE在琥珀酰胆碱中毒危象的治疗中有潜在应用前景。

Purification of genetically engineered human butyrylcholinesterase and detoxification of succinylcholine in vitro

AIM To develop a plenty and secure resource of human butyrylcholinesterase, and examine its detoxification efficacy. METHODS The purification of recombinant human butyrylcholinesterase(rhBChE) was based on the fractionation of the hemolymph proteins of silkworm larvae by ammonium sulfate and the procainamide-Sepharose 4B affinity chromatography in batches. RESULTS The overall yield of the SDS-PAGE pure rhBChE activity came to 66% of the hemolymph. The specific enzyme activity of the purified rhBChE reached at 715 mmol*min-1*g-1 protein. The catalytic activity of the enzyme stored in 20% glycerol, 0.02% NaN3, 10 mmol*L-1 sodium phosphate buffer (pH 7.4) at -20℃ for one year showed little change. Mice challenged ip with 1.5 LD of succinylcholine preincubated with rhBChE survived (18/18) without any symptom and sign of intoxication, whereas the mice in the succinylcholine control group all (18/18) died of convulsion and suffocation within 2-5 min. CONCLUSION The methods used are valid for purification of rhBChE from the hemolymph of the infected larvae. The genetically engineered rhBChE is of potential use in the therapy of succinylcholine crisis.