摘要
目的:制备高纯度的肠三叶因子(ITF),研究其理化性质及其在模拟胃肠道环境中的稳定性.方法:首先利用硫酸氨分级沉淀、镍离子亲和层析、超滤离心三步法纯化ITF,然后分析氨基酸序列、肽指纹图谱、三级结构,最后建立模拟胃肠道环境模型,研究ITF的稳定性.结果:经过三步纯化后ITF的纯度超过95%,产量约为50mg/L;氨基酸序列分析、肽指纹图谱分析与预测值完全一致,质谱分析显示精确Mr约为7879.4,与理论值(7679.7)基本一致;三级结构研究证实ITF主要以二聚体的形式存在;模拟胃肠道环境中的稳定性研究显示,ITF在蛋白酶/ITF的比例高达1∶1时,经过4h消化,ITF二聚体残余量超过50%;在pH值2.0～12.0的缓冲液中,37℃水浴5h后,残余量均超过90%;pH7.8缓冲液37℃水浴5d,残余量超过70%.结论:获得了高产量、高纯度及高度稳定的ITF,为ITF大规模的工业化生产及口服药的制备打下了坚实的基础.
AIM： To obtain intestinal trefoil factor （ITF） with high purity, to explore the physical properties of purified ITF and to assay the stability in an in vitro simulated gastrointestinal environment. METHODS： ITF was isolated from the Pichia pastoris culture supernatant by ammonium sulfate precipitation, Ni-NTA affinity chromatography and uhrafiltration. Mass spectrometry analysis, N-terminal amino acid sequence analysis, peptide mass fingerprint analysis and non-reducing Tricine SDS-PAGE analysis were performed. An in vitro restitution model was to be established and the stability in simulated gastrointestinal environment to be determined. RESULTS： The yield of ITF was about 50 mg/ L, with the purity after purification above 95%. The N-terminal sequence of ITF was shown to be consistent with the sequences we designed. Mass spectrometry analysis demonstrated that the molecular weight of ITF was 7879.4, approximately the same as the calculated value, 7 679.7. Native ITF existed mainly in dimer form. ITF was robustly resistant to trypsin and pepsin, and even at a high enzyme/substrate （w/w） ratio of 1/1, more than half of ITF existed in dimer form. Different pH values ranging from 2.0 to 12.0 had little effect on the dimer of ITF and residual dimer was greater than 90%. After water bath at 37℃ for 5 d, more than 70% of ITF was not hydrolyzed. CONCLUSION ： We have produced soluble ITF with a high yield and high purity and with resistance to proteases, heat, acid and alkali. The results lay a foundation for the commercial production of ITF and provides theoretical evidence for the oral use of ITF.
出处
《第四军医大学学报》
北大核心
2009年第6期497-500,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30200294)
关键词
肠三叶因子
纯化
药物稳定性
intestinal trefoil factor
purification
drug stability
作者简介
孙勇．硕士生（导师彭毅志），主治医师．Tel：（0516）83349405 Email：sunyong_97@sina．com
通讯作者：彭曦．Tel：（023）68754148Txt8056 Email：pxlrmm@163．com
