用色谱法纯化和鉴定牛血清白蛋白
Purification and Characterization of Bovine Serum Albumin Using Chromatographic Method.
作者信息
Balkani Sanaz, Shamekhi Sara, Raoufinia Ramin, Parvan Reza, Abdolalizadeh Jalal
机构信息
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; University of Tehran Kish International Campus, Tehran University, Tehran, Iran.
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
出版信息
Adv Pharm Bull. 2016 Dec;6(4):651-654. doi: 10.15171/apb.2016.080. Epub 2016 Dec 22.
Albumin is an abundant protein of blood and has many biopharmaceutical applications. The aim of this study was to purify bovine serum albumin (BSA) using produced rabbit anti-BSA antibody. The polyclonal antibody was produced against the BSA in rabbits. Then, the pure BSA was injected to three white New Zealand rabbits. ELISA test was done to evaluate antibody production. After antibody purification,the purified antibody was attached to CNBr-activated sepharose and finally it was used for purification of albumin from bovine serum. Western blotting analysis was used for functional assessment of immunoaffinity purified BSA. The titer of anti-bovine albumin determined by ELISA was obtained 1: 256000. The SDS-PAGE showed up to 98% purity of isolated BSA and western blotting confirmed the BSA functionality. Purified bovine serum albumin by affinity chromatography showed a single band with molecular weight of 66 KDa. Affinity chromatography using produced rabbit anti-BSA antibody would be an economical and safe method for purification of BSA.
白蛋白是血液中一种丰富的蛋白质，具有许多生物制药应用。本研究的目的是使用制备的兔抗牛血清白蛋白（BSA）抗体纯化牛血清白蛋白（BSA）。针对牛血清白蛋白在兔体内制备多克隆抗体。然后，将纯牛血清白蛋白注射到三只新西兰白兔体内。进行酶联免疫吸附测定（ELISA）试验以评估抗体产生情况。抗体纯化后，将纯化的抗体连接到溴化氰活化的琼脂糖上，最后用于从牛血清中纯化白蛋白。蛋白质免疫印迹分析用于免疫亲和纯化的牛血清白蛋白的功能评估。通过ELISA测定的抗牛白蛋白效价为1:256000。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示分离的牛血清白蛋白纯度高达98%，蛋白质免疫印迹证实了牛血清白蛋白的功能。通过亲和色谱纯化的牛血清白蛋白显示出一条分子量为66千道尔顿的单一谱带。使用制备的兔抗牛血清白蛋白抗体进行亲和色谱将是一种经济且安全的纯化牛血清白蛋白的方法。
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