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杂色鲍血蓝蛋白多克隆抗体的制备与血蓝蛋白35kDa片段鉴定
姜敬哲¹, 韩焘^1,2, 王江勇¹, 杨慧英^1,2, 刘金叶^1,2
1.中国水产科学研究院南海水产研究所;2.上海海洋大学

摘要:

以杂色鲍(Haliotis diversicolor Reeve)为研究对象, 通过密度梯度离心方法纯化得到高纯度血蓝蛋白, 以其作为抗原皮下注射免疫新西兰大白兔, 从而获得高效价的兔源多克隆抗血清。进一步通过Protein A 抗原亲和纯化的方法对该抗血清纯化, 最终获得效价更高、检测特异性更好的血蓝蛋白多克隆抗体。应用该抗体进行Western 检测发现, 鲍血淋巴中存在着多样的血蓝蛋白衍生产物; 进一步结合质谱技术对其中35 kDa 条带进行鉴定发现, 其来源于血蓝蛋白I 型亚基的H 结构域。

关键词: 杂色鲍(Haliotis diversicolor Reeve) 血蓝蛋白亲和纯化多克隆抗体 35 kDa 蛋白

DOI：

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基金项目:现代农业产业技术体系建设专项资金(CARS-48); 中央级公益性科研院所基本科研业务费专项资金项目(2010TS16); 广东省海洋渔业科技推广专项(2008899E01)

Preparation of hemocyanin polyclonal antibody and the identification of 35 kDa hemocyanin fragment in Haliotis diversicolor Reeve

Abstract:

Haliotis diversicolor Reeve, was taken as research material. High purity hemocyanin proteins were purified from the hemolymph of abalone using CsCl density gradient centrifugation method. By injection of the purified antigen to a rabbit, high titer polyclonal anti-serum was acquired. The anti-serum was further processed with the protein A affinity purification to produce purified antibodies. Finally, the purified polyclonal antibodies with higher titer and specificity were acquired. Further Western blotting with these antibodies, plenty of hemocyanin was found as derivates of various molecular weights in abalone hemolymph.

Key words: Haliotis diversicolor Reeve hemocyanin affinity purification polyclonal antibody 35k Da protein.

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