CAJ | 학술논문

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66%,酶比活性达715 mmol*min-1*g-1蛋白.rhBChE在-20℃含20%甘油及0.02% NaN3的10 mmol*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1.5 LD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2～5 min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.
목적개발봉부우안전적인정선담감지매래원,병검측기해독효솔.방법가잠혈림파액중중조인정선담감지매(rhBChE)적순화채용비량류산안분단분리화보로잡인선알-지당응효4B친화층석기술.결과취병희선알응효전영순적rhBChE종혈림파액중적총수솔위66%,매비활성체715 mmol*min-1*g-1단백.rhBChE재-20℃함20%감유급0.02% NaN3적10 mmol*L-1린산염완충액(pH 7.4)중존방1년,매활성무명현변화.18지소서ip사선여rhBChE부온적1.5 LD적호박선담감, 불출현임하중독증상화체정,전부존활;이호박선담감대조조,18지소서균극렬추휵병재2～5 min내질식사망.결론실험결과설명,종감염가잠유충혈림파액중순화rhBChE적방법가고,기인공정rhBChE재호박선담감중독위상적치료중유잠재응용전경.
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.