福建产眼镜王蛇毒神经毒素的分离及活性测定

The Isolation and Charaterization of the Neurotoxic Fractions from Ophiophagus hannah Venom

目的 从眼镜王蛇蛇毒中分离神经毒素 ,测定其活性和毒性。 方法 应用 CM- Sephadex C- 5 0离子交换色谱 ,阶段梯度洗脱分离粗毒 ;用 Sephadex G- 5 0凝胶过滤纯化组分 ;用大鼠体外膈神经 -膈肌标本鉴定分离组分对神经 -肌肉接头的作用性质 ;观察神经毒素对乙酰胆碱 (Ach)引起蛙体外腹直肌收缩的量效曲线的影响 ;以Meier法测定神经毒素对小鼠的半数致死量 (L D50 )。 结果 眼镜王蛇粗毒经阳离子交换色谱后获得 A～ N共 1 4个蛋白峰 ,经鉴定 E、F、G、H、I、K等 6个蛋白峰具有阻断神经 -肌肉接头传导的作用 ,被鉴定为神经毒素 ,约占粗毒的 4 8.3% ;通过凝胶过滤对 E、G和 K组分进行初步纯化 ;E、G和 K组分可使 Ach引起的蛙腹直肌收缩的量效曲线平行右移 ,它们与 N2 -胆碱受体 (N2 - Ach R)的解离常数 (KD)分别为 :1 4 5 .87,73.5 3和 2 0 .0 4 ng/ m L;E、G和 K组分小鼠静脉注射的 L D50 分别为 :0 .884 ,0 .74 9和 1 .5 8mg/ kg。 结论 眼镜王蛇粗毒经离子交换色谱获得 6个神经毒素组分 ,经鉴定 E、G和 K组分为 N2 - Ach

Objective To isolate and charaterize the neurotoxic fractions from Ophiophagus hannah venom. Methods CM-Sephadex C-50 column was used to isolate the venom with step-wise elution. The fractions were purified by Sephadex G-50. Neurotoxicity was determined on isolated rat phrenic nerve-diaphragm preparations. Effects of neurotoxic fractions on acetylcholine-induced contractile response of the isolated frog rectus abdominis were observed. The LD-{50} of neurotoxic fractions was determined by Meier and Theakston method. Results Forteen fractions(A^N) were isolated from Ophiophagus hannah venom, six fractions(E, F, G, H, I and K) of which are neurotoxic and occupied the content of 48 3%. Fraction E, G and K produced a concentration-dependent parallel shift to the right of dose-response curve of acetylcholine applied exogenously with no change in the maximal asymptotic response. The dissolation constant K-D of N-2-AchR in fraction E, G and K were 145 87, 73 53 and 20 04 ng/mL, respectively. The LD-{50} of fraction E, G and K in mice(iv) were 0 884, 0 749 and 1 58 mg/kg respectively. Conclusion Six neurotoxic fractions were isolated from Ophiophagus hannah venom(from Fujian). Three fractions(E, G and K) were determined as the competitive antagonists of N-2-AchR.;