关键词： 中华眼镜蛇毒，神经生长因子，鉴定

摘要：目的：控制中华眼镜蛇蛇毒神经生长因子产品质量，研究其理化性质及生物学活性的定性和定量。方法：通过离子交换色谱、凝胶过滤及FPLC色谱分离纯化得到中华眼镜蛇蛇毒神经生长因子，按国家新药审批有关要求对其进行了SDS-PAGE电泳，N端蛋白质序列测定，HPLC色谱分析，UV光谱图谱扫描，并利用PC12细胞培养法和鸡胚背根神经节培养法检测其生物活性。结果：电泳为一条带，亚基分子量为13500，N端蛋白质序列测定后确证为神经生长因子(NGF)，HPLC为单峰，相对百分含量为95％以上，279.6nm处呈现出蛋白质样特征吸收峰。生物活性测定为，PCl2细胞培养法灵敏度可达lng／ml，鸡胚背根神经节培养法需30ng／ml的浓度梯度才能在神经节上有所反应。结论：此实验样品为具有较高生物活性的高纯度NGF多肽。

分类号：R973.2

Studies on Bioassay and Properties of Nerve Growth Factor from Naja Naja Atra Venom

dong Yuewei,Xu Tianrui,Xiong Yuliang,Xu Kangsen(Yunnan Provincial Institute for Drugs Control Kunming 650011)

Abstract：Purpose: To control quality of nerve growth factor,its bioassay and properties were studied. Methods: According to the requirements of new drugs, the NGF monomer molecular weight was measured by SDS-PAGE and its partial N-terminal sequence was delermined by 476A peptide/protein sequencer. The purity was tested by HPLC, UV spectrum scanning and SDSPAGF. Two bioassay methods including chicken embryo dorsal ganglion culture and PC12 cell culture were applied to assay the bioactivity. Results:The NGF monomer molecular weight was estimated to be 13 500 and its partial N-terminal sequence was consistent with other NGF isolated from the same genus. NGF had a specific absorbing peak at 279.6nm in UV spectrum and showed a homogeneous band in SDS-PAGE and HPLC. Bioassay experiment showed that the greatest outgrowth of chicken embryo dorsal ganglion culture evoked by NGF at a concentration of 30ng/ml,but in the PC12 cell culture its concentration was only lng/ml. Conclusion:The tested sample is a purified NGF with higher bioactivity.

Keywords：Naja naja atra venom, Nerve growth factor, Characterization基金项目：云南省重点基金项目(980468)参考文献：

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收稿日期：1999-07-12

修稿日期：1999-12-30