猪膜联蛋白A2多克隆抗体的制备、亲和纯化与鉴定  

Preparation,Affinity Purification and Identification of Polyclonal Antibody Against Pig Annexin A2

作　　者：陈江涛[1] 王光祥[1] 吉艳红[1] 尚佑军[1] 刘艳红[1] 刘湘涛[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室国家口蹄疫参考实验室农业部畜禽病毒学重点开放实验室,兰州730046

出　　处：《畜牧兽医学报》2010年第11期1502-1509,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金："十一五"国家科技支撑计划重大项目(2006BAD06A03)

摘　　要：本研究旨在制备并亲和纯化兔抗猪膜联蛋白A2多克隆抗体。通过PCR从质粒pA2-EGFP中将编码猪膜联蛋白A2与增强型绿色荧光蛋白融合基因(Annex A2-EGFP)亚克隆到pET-30a(+),构建了原核表达质粒p30a/AE,转化大肠杆菌Rosetta(DE3),经IPTG诱导以包涵体形式表达了猪ANNXA2-EGFP融合蛋白,用Ni-Resin HP树脂对表达的目的蛋白进行亲和纯化。用纯化的ANNXA2-EGFP融合蛋白免疫兔,制备兔抗猪膜联蛋白A2多克隆抗体。将以前纯化的猪可溶性GST-ANNXA2融合蛋白偶联NHS活化琼脂糖凝胶FF,亲和纯化多克隆抗体。用GST-ANNXA2亲和纯化的多抗效价达到1∶12800,Western blot和免疫组化结果证实具有高特异性。制备的高效价和高特异性兔抗猪膜联蛋白A2多克隆抗体,为进一步研究膜联蛋白A2在病毒感染中的作用奠定了基础。The study was aimed at preparing and purifying polyclonal antibody of rabbit anti-pig ANNXA2 by GST-ANNXA2-coupled affinity chromatography.The fragment of pig Annexin A2 and EGFP fusion gene was amplified from pA2-EGFP plasmid by PCR.And then the fragment was subcloned into the prokaryotic expression vector pET-30a（＋） to construct the recombinant plasmid p30a/AE,and expressed in inclusion body of E.coli.Rosetta（DE3） by IPTG induction.The fusion protein ANNXA2-EGFP which was purified by Ni-Resin HP affinity chromatography was used as immunogen to inject male rabbit to prepare polyclonal antibody anti-pig Annexin A2.The soluble fusion protein GST-ANNXA2 which has been prepared was coupled on NHS-activated Sepharose Fast Flow column,and then the polyclonal antibody was purified by this column.Purified antiserum titer amounted to 1：12 800 by indirect ELISA,and the purified antiserum was also demonstrated high specificity by Western blot and immunohistochemistry staining.The polyclonal antibody of rabbit anti-pig ANNXA2 was prepared and possessed high titer and high specificity,which laid the groundwork for the investigation of relationship between virus infection and pig Annexin A2.

关 键 词：猪膜联蛋白A2 多克隆抗体 亲和纯化 

分 类 号：S852.23[农业科学—基础兽医学]