一种分离提纯眼镜蛇(Naja naja atra)神经毒素的简便方法

A SIMPLE METHOD FOR ISOLATION AND PURIFICATION OF COBRA NEUROTOXIN FROM THE VENOM OF NAJA NAJA ATRA

本文报导了一种分离提纯眼镜蛇神经毒素的简便方法:用pH7.8,0.01M磷酸盐缓冲液平衡的CM-纤维素柱吸附眼镜蛇毒,经平衡缓冲液洗涤后,以含不同NaCl浓度的平衡缓冲液分段洗脱。眼镜蛇神经毒素在用含0.07M NaCl的缓冲液洗脱时被洗脱下来,此组份的洗脱液合并后经70℃选择性热变性处理,冷却后离心除去变性的杂蛋白沉淀,溶液加入三倍体积的冷丙酮,得到含盐的眼镜蛇神经毒素沉淀,用Sephadex G10脱盐后冻干,即可得到纯眼镜蛇神经毒素。此法分离提纯的眼镜蛇神经毒素,聚丙烯酰胺凝胶电泳显示出单一的区带,Sephadex G-50凝胶过滤层析出现单一的、对称的洗脱峰,与抗眼镜蛇毒血清进行免疫扩散时呈现单一的沉淀线。此神经毒素小鼠腹腔注射的LD;为1.1ug/20g体重,当浓度为1.Omg/ml时,A;为1.07。得率约为全毒的6.5%。

A simple method for isolation and purification of cobra ncurotoxin from the venom of Naja naja atra was reported.The cobra venom was absorbed on CM-cellulose pre-equilibrated with pH 7.8,0.01M phosphate buffer.The stepwise elution was carried out with equilibrium buffer containing several concentrations of sodium chloride after washing with equilibrum buffer.Cobra neurotoxin was eluted with equilibrium buffer containing 0.07 NaCl.This pooled fraction was treated with selective heat-denaturation at 70.C.Denatured protein was removed by centriiugation.The cobra ncurotoxin was precipitated with 3 volumes of cold acetone from the solution.The purified cobra neurotoxin was obtained after desalted on Sephadex G-10 and lyophilized.Cobra neurotoxin purified by above described method showed a single band on electrophorcsis in polyacrylamide gel.A symmetrical cluted peak was exhibited by gel filtration on Sephadex G-50.It showed a single precipitate band when the cobra neurotoxin reacted with anti-cobra venom antisera.LD50(i.p.)of the purified cobra neurotoxin was 1.1 ug/20g oi mouse.When the concentration of cobra neurotoxin was 1.0 mg/ml,A1cm/280nm=1.07.The yield oi the purified cobra neurotoxin was 6.5%approximately.