人心脏型脂肪酸结合蛋白的纯化与鉴定  

Isolation and identification of heart-type fatty acid-binding protein from human myocardium

作　　者：季鹏[1] 顾春荣[1] 卞智萍[1] 陈相健[1] 张寄南[1] 杨笛[1] 

机构地区：[1]南京医科大学第一附属医院心血管病研究所,江苏省南京市210029

出　　处：《中国组织工程研究与临床康复》2007年第27期5387-5390,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30570745);江苏省政府"135"重点实验室科研基金(SK200205)~~

摘　　要：目的:以往的心脏型脂肪酸结合蛋白多来自牛、鼠等动物心肌组织,本实验构建了人左心室心肌组织纯化心脏型脂肪酸结合蛋白的新方法。方法:实验于2005-01/03在南京医科大学第一附属医院心血管病研究所完成。①实验材料:人心室肌由南京医科大学第一附属医院心血管病研究所提供。②实验过程:取20g人左心室肌在缓冲液中匀浆,多步骤离心后以(NH4)2SO4进行盐析→再次离心→沉淀缓冲液重悬透析得蛋白粗提液→Sephadex G-75凝胶过滤→电泳确定主要蛋白条带的收集管号合并收集→QSepharose Fast Flow阴离子交换层析→聚乙二醇浓缩→缓冲液透析得人心脏型脂肪酸结合蛋白。③实验评估:以SIGMA公司人心脏型脂肪酸结合蛋白为对照标准品;以Tricine-SDS-PAGE电泳鉴定相对分子质量;以鼠抗人脂肪酸结合蛋白单克隆抗体及羊抗鼠IgG对其进行Western印迹检测鉴定其特异性。结果:①Sephadex G-75凝胶过滤柱层析结果:各组分经Tricine-SDS-PAGE电泳检测证实,部分收集管中存在相对分子质量约14400的人心脏型脂肪酸结合蛋白。②阴离子交换柱层析结果:在洗脱液NaCl浓度达6mmol/L时,出现蛋白峰,人心脏型脂肪酸结合蛋白被洗脱;NaCl浓度达20mmol/L时,蛋白峰降至基线,蛋白洗脱结束。③纯化的人心脏型脂肪酸结合蛋白鉴定结果:经电泳显示其相对分子质量约为14000,纯度约为90%;Western印迹证实其可被抗人脂肪酸结合蛋白单克隆抗体特异识别,与对照标准品SIGMA公司人心脏型脂肪酸结合蛋白结果一致。实验从20g湿重心室肌中共纯化得到15.4mg心脏型脂肪酸结合蛋白,得率为0.77mg/g。结论:Sephadex G-75凝胶过滤柱层析及阴离子交换柱层析相结合,可用于人心脏型脂肪酸结合蛋白的纯化。AIM： The heart-type fatty acid-binding protein （H-FABP） is derived from the myocardial tissue of cow, rat and other animals before. In this study, a new method to isolate H-FABP from human left ventricles was established. METHODS： The experiment was performed in the Institute of Cardiovascular Disease, First Affiliated Hospital of Nanjing Medical University from January to March 2005. 020 g human myocardium （Institute of Cardiovascular Disease, First Affiliated Hospital of Nanjing Medical University） were dissected and homogenized in PBS buffer. The mixture was centrifuged and the protein was salted out with （NH4）2SO4. Then the supernatant was collected and centrifuged again, The sediments were re-suspended and dialyzed with PBS buffer before being subjected to Sephadex G-75 gel filtration. Combined protein fractions were separated by Tricine-SDS-PAGE in which the band mainly distributed in 14.4 KD was estimated, and then the peak proteins were collected by subjecting to anion-exchange chromatography. The H-FABP protein was obtained after condensed with polyethylene glycol （PEG） and dialyzed with stock buffer. （2）The purified protein was identified by Tricine-SDS-PAGE and Western-blot referring to the standard H-FABP protein obtained from SIGMA, RESULTS： （1）The molecular mass of the isolated protein detected with Tricine-SDS-PAGE was approximately Mr 14 400. （2）The peaks of protein appeared and H-FABP was eluted when the concentration of eluant NaCl reached 6 mmol/L; the peak of protein declined to the baseline and protein elution was over when the concentration reached 20 mmol/L with Anion-exchange chromatography. （3）Identification of purified H-FABP： The relative molecular mass of H-FABP was about 14 000, and the purity was about 90%. Western blot proved that the purified H-FABP had specific binding affinity to anti-H-FABP monoclonal antibody, which was in accordance with the standard H-FABP protein obtained from SIGMA, Totally 15.4 mg H-FABP was obtained from 20 g （wet

关 键 词：载体蛋白质类 人 分离和提纯 组织构建 

分 类 号：R341[医药卫生—基础医学]