人血浆脂联素分离纯化及活性鉴定

廉坤; 王海昌; 王汝涛; 靳温; 孙璐; 马新亮; 陶凌

人血浆脂联素分离纯化及活性鉴定

Isolation and purification of adiponectin multimers from human plasma and detection of their bioactivity

摘要

摘要: 目的: 从人血浆中分离纯化各亚型脂联素(APN)，建立人血浆APN活性的检测方法,同时获得制备抗APN各亚型特异性抗体所需的抗原。方法：将-80℃保存的人血浆融解后稀释，采用硫酸铵沉淀、阴离子交换层析和凝胶过滤层析分离纯化人血浆中不同亚型的APN。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫印迹（Western blot）和ELISA检测纯化的产物及回收率。通过孵育人脐静脉内皮细胞后，检测磷酸腺苷激活的蛋白激酶（AMPK）的水平反映APN的活性。结果：所获不同亚型的APN均具有生物学活性，高聚体APN激活AMPK的能力最强。APN活性回收率为40%。结论：建立了一种能够快速、简便从人血浆中分离纯化APN及其亚型并保持其天然的生物学活性的方法，为APN的深入研究奠定了基础。

Abstract: AIM: To isolate adiponectin isoforms from human plasma, establish a method to detect human plasma adiponectin isoform biological activity and provide antigen to prepare adiponectin isoform-specific antibody. METHODS: Human plasma stored at -80℃ was thawed and diluted. Plasma adiponectin multimers were isolated by ammonium sulfate precipitation, anion-exchange chromatography and gel filtration chromatography. Adiponectin purity and recovery rate were detected by polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and enzyme-linked immunosorbent assay (ELISA). Adiponectin bioactivity was detected by incubating human umbilical vein endothelial cells with purified adiponectin isoform and determining the phosphorylation level of AMP-activated protein kinase. RESULTS: The three adiponectin isoforms isolated from plasma all activated AMPK and the high-molecular-weight form was the most potent. Adiponectin activity recovery rate was about 40%. CONCLUSION: Our method efficiently purifies adiponectin multimers from human plasma and preserves their bioactivity, which establishes the basis for further study on adiponectin.

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