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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。
Objective To develop a rich and safe source of human butyrylcholinesterase and test its detoxification efficiency. Methods Purification of recombinant human butyrylcholinesterase (rhBChE) in the hemolymph of silkworm (Bombyx mori) using batch ammonium sulfate fractionation and procaine amide glycolipid 4B affinity chromatography. Results The total yield of rhBChE from polyacrylamide gel electrophoresis was 6 6% and the specific activity of enzyme was 715 mmol · min -1 · g -1. rhBChE was stored for 1 year in 10 mmol·L-1 phosphate buffer (pH 7.4) containing 20% glycerol and 0.2% NaN3 at -20 ° C without any significant change in enzyme activity. Eighteen mice ip pre-incubated with rhBChE 1.5LD of succinylcholine, did not show any signs and symptoms of poisoning, all survived; and succinylcholine control group, 18 mice were severe convulsions and 2 ~ 5min Asphyxiation death. Conclusion The experimental results show that the method of purifying rhBChE from the hemolymph of infected silkworm larvae is reliable, and the genetically engineered rhBChE has potential application prospect in the treatment of succinylcholine poisoning crisis.