利用Pen a 1表位抗体亲和纯化目标蛋白的研究  

Purification of Pen a 1 Using Immunoaffinity Column of Polyepitope Antibody Against Pen a 1

作　　者：赵杰[1] 高美须[1] 潘家荣[2] 兰丽平[1] 睢珂[1] 牟慧[1] 许舒婷[1] 

机构地区：[1]中国农业科学院农产品加工研究所,北京100193 [2]中国计量学院生命科学院,浙江杭州31008

出　　处：《分析测试学报》2013年第10期1174-1179,共6页Journal of Instrumental Analysis

基　　金：国家"十二五"科技支撑课题(2011BAK10B03);农业部公益性行业科研专项(201103007)

摘　　要：以虾致敏蛋白Pen a 1（Tropomyosin）抗原表位为研究对象,建立了利用Pena 1表位抗体亲和纯化致敏蛋白的新方法.Fmoc法合成致敏Pen a 1蛋白的C端含有3个抗原表位的第247 ～284位氨基酸对应的多肽片段,应用马来酰亚胺法将多肽与KLH（匙孔血蓝蛋白）、BSA（牛血清白蛋白）偶联制备人工免疫抗原（Pep-tide-KLH）和人工包被抗原（Peptide-BSA）,免疫人工抗原免疫纯种新西兰白兔,获得多克隆抗血清,抗血清经辛酸-硫酸铵及特异性血清纯化预装柱（HiTrap rProtein A FF）纯化后与溴化氰活化琼脂糖凝4B（CNBr-Activated Sepharose 4B）进行偶联.ELISA（酶联免疫吸附试验）测定该多克隆抗体效价为2.05×106,多肽对抗体的IC50（50％抑制浓度）为0.21 mg/L,交叉试验表明该抗体与虾中非Pena 1蛋白无交叉反应性;Bradford法测定CNBr-Activated Sepharose 4B与抗体的偶联率为90.76％.间接竞争ELISA测定1 mL偶联介质的吸附容量为2.84 mg Pen a 1,免疫亲和柱的加标回收率为89.6％～93.6％,亲和柱使用寿命为4次.A new method for the purification of shrimp allergenic protein Pen a 1 was developed.The epitope peptide （aa 247 to 284） of the Pen a 1 was synthesized by standard Fmoc and conjugated to KLH and BSA to get the artificial immune antigen （peptide-KLH） and the coating antigen （peptide-BSA）,respectively.New Zealand white rabbits were immunized with immunogen peptide-KLH to produce antisera and the antisera were purified by bitterness-ammonium sulfat and HiTrap rProtein A FF.The pure antiserum was coupled to CNBr-Activated Sepharose 4B.The titer of purified antibody was 2.05 × 106 by indirect ELISA and the IC50 was 0.21 mg/L by indirect competitive ELISA method.There was no cross-reactivity between the antibody and the proteins from shrimp except for Pen a 1 by indirect competitive ELISA method.The coupling efficiency of the antibody with CNBr-Activated Sepharose 4B was 90.76％ and the column capacity for 1 mL immunosorbents was 2.84 mg Pen a 1.The recoveries of the immunoaffinity column were in the range of 89.6％-93.6％.The service life of the immunoaffinity column was 4 cycles.The SDS-PAGE and Western Blot results displayed that the purified Pen a 1 had high immunogenicity.

关 键 词：PEN a 1 表位抗体 免疫亲和柱 ELISA 

分 类 号：O766.1[理学—晶体学] TQ424.27[化学工程]