A novel peptides, like nerve growth factor, inducing pheochromocytoma PC12 cell differentiation

摘要

目的:根据NGF的氨基酸序列及其晶体构象资料,对NGF-β进行限制性酶解,从而获得关键的功能区域片段.方法:选择CNBr在9位Met处, Trypsin在Arg或Lys处裂解NGF-β,用 Sephadex G50层析、DE52离子交换层析和反相高压液相层析进行分离纯化.所得肽片段用PC12细胞测定活性,对活性片段进行氨基酸组成分析及氨基酸序列分析.结果:从NGF-β裂解片段中获得一可诱导PC12细胞分化的活性片段,氨基酸组成分析及氨基酸序列分析此片段由16肽GEFSVCDSVSVWVGDK与14肽HWNSYCTTTHTFVK通过一对二硫键连接而成,与NGF-β肽链的10～25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0.001～0.1 μg*L-1.结论:本实验获得了NGF-β关键的功能区域片段.虽然其空间结构和其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关键的基础.%Objective: To isolate the key domain of a novel polypeptide fragment from NGN-β that functions like intact NGF molecule.Methods: NGF-β had been treated with cyanogen bromide and trypsin skillfully. The peptide fragment with the activity inducing PC12 pheochromocytoma cells differentiation was isolated and purified by Sephadex G-50 gel filtration chromatography , DE-52 celluloseion exchange chromatography and C-18 reversed-phase column HPLC after NGF-β cleavaged by CNBr at 9th met then by trypsin at Arg or Lyscleavaged by CNBr at 9th met then by trypsin at Arg or Lys. Amino acid sequencing of this novel peptide fragment was performed by Automatic Amino Acid Analyser and Amino Acid Sequencer. Results: The functional fragment from cleavaged NGF might induce differentiation of PC12 cells. The fragment was consisting of two linear polypeptides . One of them was 16 peptide, GEFSVCDSVSVWVGDK , and other was 14 peptide, HWNSYCTTTHTFVK, linked by a disulphide bridge corresponding to residues 10～25 and 75～88, respectively, of the amino acid sequence of nerve growth factor, the result of biological activity assay in PC12 cells showed that the optimum concentration of this peptide were 0.001～0.1 μg*L-1. Conclusion: A novel peptide inducing differentiation of PC12 cell line of pheochromocytoma cells was obtained in the study. It′s isolation and purification successfully will underlie synthesis or expression of hyperactive neurotrophic small molecular substance although the relationship between the configuration and functions is not clearly.