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目的 :研究浙江眼镜蛇 (Najanajaatra)蛇毒的镇痛活性组分 ,为寻找镇痛效果好 ,而无成瘾性的新型镇痛药奠定基础。方法 :通过两次CM SephadexC 2 5和一次SephadexG 5 0柱层析对眼镜蛇毒进行了分离纯化。采用PAGE法对分离纯化后的眼镜蛇神经毒素 (cobroneurotoxin ,CNT)纯度进行鉴定 ;以SDS PAGE法测定CNT的分子量 ;等电聚焦法 (Isoelectrofo cusing ,IEF)测定CNT的等电点 ;以Lowry法测定其蛋白含量 ;用醋酸扭体法、热板法与电刺激法考察CNT的镇痛活性并通过测定LD50 考察其毒性。结果 :眼镜蛇毒经 3次柱色谱后 ,CNT经鉴定为单一组分。经SDS PAGE法测定其分子量为 70 0 0 ,等电点为 10 .2 ,蛋白含量为 92 %。CNT能显著降低小鼠对化学刺激与电刺激的敏感性 ;小鼠痛阈百分率的提高可达 5 1.3% ,并呈一定的量效关系 ;小鼠腹腔注射的LD50 为 81μg·kg-1,95 %可信限为 5 8～ 10 4 μg·kg-1。结论 :眼镜蛇毒经 3次柱色谱后得到具有镇痛活性的单一组分 (CNT)。
Objective: To study the analgesic active ingredients of Najanajaatra venom from Zhejiang, and to lay a foundation for finding new analgesics with good analgesic effect and no addiction. Methods: Cobra venom was isolated and purified by two CM Sephadex C 2 5 and one SephadexG 50 column chromatography. The purity of coboneurotoxin (CNT) was isolated and purified by PAGE. The molecular weight of CNTs was determined by SDS PAGE, the isoelectric point of CNT was determined by isoelectric focusing (IEF) The protein content was determined by acetic acid writhing method, hot plate method and electrical stimulation method. Results: Cobra venom was identified as a single component after three column chromatography. The molecular weight was 70 0 0, the isoelectric point was 10. 2 and the protein content was 92% as determined by SDS PAGE. CNT can significantly reduce the sensitivity of mice to chemical stimulation and electrical stimulation; the mouse pain threshold percentage increased up to 53.3%, and showed a dose-effect relationship; LD50 of mice injected intraperitoneally was 81μg · kg-1, The 95% confidence limits ranged from 58 to 104 μg · kg-1. Conclusion: The cobra venom obtained single component (CNT) with analgesic activity after three column chromatography.