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目的根据神经生长因子 (NGF)的氨基酸序列及其晶体构象资料 ,对 β NGF进行限制性酶解 ,从而获得关键的功能区域片段。方法选择溴化氰在 9位Met处 ,Trypsin在Arg或Lys处裂解 β NGF ,用SephadexG 5 0色谱、DE5 2离子交换色谱和反相高效液相色谱进行分离纯化。所得片段用PC12细胞测定活性 ,对活性片段进行氨基酸组成及序列分析。结果从 β NGF裂解片段中获得一可诱导PC12细胞分化的活性片段 ,氨基酸组成及序列分析表明 ,此片段由 16肽GEFSVCDSVSVWVGDK与 14肽HWNSYCTTTHTFVK通过一对二硫键连接而成 ,与 β NGF肽链的10～ 2 5和 75～ 88氨基酸残基序列相对应。生物活性分析表明其最佳作用浓度为 0 .0 0 1～ 0 .1ng/ml。结论此实验获得了 β NGF关键的功能区域片段 ,虽然其空间结构和功能的关系尚需研究探讨 ,但它的成功分离和鉴定为合成或表达高活性小分子神经营养物质奠定了重要的基础
OBJECTIVE: According to the amino acid sequence of nerve growth factor (NGF) and its crystal conformation, β NGF was restricted by enzymatic hydrolysis to obtain the key functional region fragments. Methods Cytotoxicity of cyanogen bromide at 9 Met was studied. Trypsin lysed β NGF at Arg or Lys, and purified by Sephadex G 50, DE 5 2 ion exchange chromatography and reversed-phase high performance liquid chromatography. The resulting fragments were assayed for activity using PC12 cells and amino acid composition and sequence analysis of the active fragments. Results A fragment that could induce the differentiation of PC12 cells was obtained from β-NGF cleavage fragment. The amino acid composition and sequence analysis showed that this fragment was composed of the 16-mer peptide GEFSVCDSVSVWVGDK and the 14-peptide HWNSYCTTTHTFVK linked by a pair of disulfide bonds, 10 to 25 and 75 to 88 amino acid residue sequences corresponding to. Biological activity analysis showed that the best concentration was 0.0101 ~ 0.1ng / ml. Conclusions The key functional region fragment of β-NGF was obtained in this experiment. Although the relationship between its spatial structure and function needs further study, its successful isolation and identification lay an important foundation for the synthesis or expression of high-activity small-molecule neurotrophic substances