小麦种子过氧化物酶wp1 基因的原核表达、纯化及多克隆抗体制备.pdf

生 物 工 程 学 报 Chin J Biotech 2011, January 25; 27(1): 26−30 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ ©2011 CJB, All rights reserved. 工业生物技术 小麦种子过氧化物酶 WP1 基因的原核表达、纯化及多克 隆抗体制备 1 1 1 1,2 1,2 单丽伟 ，唐如春 ，刘三阳 ，范三红 ，郭蔼光 1 712100 2 712100 摘 要: WP1 是小麦种子中最主要的阳离子过氧化物酶，该酶不仅参与种子的发育过程，而且影响面粉的加工品质。 首先构建了 WP1 基因原核表达载体pET28a-WP1 ，并将其转化到T7 Expression 大肠杆菌菌株中诱导表达。His-tag 融合 的WP1 主要以包涵体形式存在，使用Ni-NTA 亲和层析柱在变性条件下进行纯化，获得纯度大于98 ％的重组蛋白。重 组WP1 经尿素梯度透析复性溶解后免疫新西兰大白兔，最终获得WP1 多克隆抗体。ELISA 分析结果显示制备的WP1 兔抗血清的效价大于 1 ∶625 000；Western blotting 结果证明制备的多克隆抗体对WP1 具有很好的专一性。 关键词: 小麦，种子过氧化物酶，WP1 ，表达纯化，抗体制备 Prokaryotic expression, purification and preparation of polyclonal antibody for wheat grain peroxidase WP1 gene 1 1 1 1,2 1,2 Liwei Shan , Ruchun Tang , Sanyang Liu , Sanhong Fan , and Aiguang Guo 1 College of Life Sciences, Northwest A F University, Yangling 712100, China 2 Key Laboratory of Agricultural Molecular Biology in Shaanxi Province, Yangling 712100, China Abstract: Wheat peroxidases 1 (WP1) is the major cationic peroxidase of wheat (Triticum aestivum) grain, which is involved in the development of seeds and an important factor to affect the final processing quality of flour. We constructed a prokaryotic expression vector pET28a-WP1, and transformed it into E. coli host strain T7 Expression. His-tag fused WP1 existed as inclusion body, and the recombinant protein was purified by Ni-NTA resin affinity chromatography under denatured condition. The purity of target protein reached 98%. The recombinant WP1 was refolded by gradient urea dialysis, then used as antigen to