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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。
WP1 is the most important cationic peroxidase in wheat seeds. The enzyme not only participates in the seed development, but also affects the processing quality of the flour. First, we constructed the prokaryotic expression vector pET28a-WP1 of WP1 gene and transformed it into E. coli strain of T7 Expression to induce expression. His-tag fused WP1 mainly exists as inclusion bodies and purified by Ni-NTA affinity chromatography under denaturing conditions to obtain a recombinant protein with a purity greater than 98%. Recombinant WP1 was refolded by urea gradient gradient dialysis and immunized New Zealand white rabbits to obtain the final WP1 polyclonal antibody. ELISA analysis showed that the titer of the prepared rabbit antiserum was greater than 1: 62 000 000; Western blotting results showed that the prepared polyclonal antibody had good specificity for WP1.