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

作　　者：单丽伟[1] 唐如春[1] 刘三阳[1] 范三红[1,2] 郭蔼光[1,2] 

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100 [2]陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《生物工程学报》2011年第1期26-30,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30300222)资助~~

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。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.

关 键 词：小麦 种子过氧化物酶 WP1 表达纯化 抗体制备 

分 类 号：S512.1[农业科学—作物学]