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目的从中华眼镜蛇粗毒中分离纯化其α-神经毒组分,并将此α-神经毒作为亲和层析的配基纯化烟碱型乙酰胆碱受体(nAChR)。方法采用凝胶过滤层析、阳离子交换层析逐步分离纯化中华眼镜蛇粗毒,通过125I-α-银环蛇毒素(αBtx)-nAChR结合抑制实验检测蛋白活性,SDS-PAGE检测蛋白的相对分子质量和纯度;亲和层析纯化nAChR,放射免疫沉淀法测nAChR活性。结果从5g中华眼镜蛇粗毒中共纯化得到约20mg眼镜蛇α-神经毒,该眼镜蛇α-神经毒有一定的125I-αBtx-nAChR结合抑制活性,其半数抑制浓度(IC50)约为αBtx的28倍。以纯化的α-神经毒制备的亲和介质,可吸附大鼠骨骼肌肌膜提取物中约84%的125I-αBtx结合活性,且此活性可以被0.2mol/L卡巴胆碱洗脱。结论用简单的液相色谱方法可从中华眼镜蛇毒中分离纯化α-神经毒,并且可以以其为配体从大鼠骨骼肌中纯化nAChR。
OBJECTIVE To isolate and purify α-neurotoxic components from the crude cobra venom of Chinese cobra, and purify the nicotinic acetylcholine receptor (nAChR) by using α-neurotoxicity as the ligand for affinity chromatography. Methods The crude drug was separated and purified by gel filtration chromatography and cation exchange chromatography. The activity of protein was determined by 125I-α-bungarotoxin-nAChR binding inhibition assay. The relative molecular mass of protein was determined by SDS-PAGE And purity; affinity chromatography purified nAChR, radioimmunoprecipitation assay nAChR activity. As a result, about 20 mg of cobra α-nerve virus was co-purified from 5 g of Chinese cobra venom, and the cobra α-neurotoxicity had a certain 125I-αBtx-nAChR binding inhibitory activity with an IC50 of about 28 times that of αBtx. Affinity media prepared with purified α-neurotoxicity adsorb about 84% of 125I-αBtx-binding activity in rat skeletal muscle sarcolemma extracts, and this activity can be eluted with 0.2 mol / L carbachol. Conclusions α-neurotoxicity can be isolated and purified from the cobra venom using simple liquid chromatography and nAChR can be purified from rat skeletal muscle using it as a ligand.