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。
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.
出处
《心脏杂志》
CAS
2011年第2期209-213,共5页
Chinese Heart Journal
基金
国家高技术研究发展计划(2009AA02Z104)
关键词
脂联素
分离纯化
活性鉴定
adiponectin
isolation
purification
bioactivity
