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

Prokaryotic expression, purification and preparation of polyclonal antibody for wheat grain peroxidase WP1 gene

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。

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 immune rabbit to prepare polyclonal antibody. The result of ELISA showed that the titer of rabbit anti-WP1 antiserum was higher than 1:625 000. The result of Western blotting demonstrated that the prepared WP1 polyclonal antibody could be used to detect WP1 with high specificity.