小麦类脱水素的表达、纯化及多克隆抗体的制备

脱水素在胚胎发育后期累积,外源脱落酸(ABA)、低温、干旱和其他一些环境条件下能诱导脱水素的产生,尽管植物在脱水条件下脱水素广泛存在于细胞中,但其生化功能仍不清楚.为研究小麦在不同时期脱水素基因的表达情况和生物学功能及抗体制备,以小麦幼芽为材料,经干旱胁迫处理后,提取总RNA,通过RT-PCR得到小麦类脱水素基因片段(WZY1-1),再连接至克隆载体PUCM-T,并成功构建重组表达质粒PET-32a( )-wzy1-1,将阳性重组质粒转化于受体菌BL21(DE3)感受态细胞中,经IPTG诱导表达,进行表达产物的聚丙烯酰胺凝胶电泳(SDS-PAGE)检测.结果表明,表达蛋白位于37ku处,小麦类脱水素基因获得高效表达.表达蛋白经Ni2 琼脂糖凝胶亲和层析和透析袋电洗脱法纯化后,对兔子进行免疫,制备的抗血清通过ELISA检测到较高的多克隆抗体效价.蛋白质印迹结果显示,利用纯化的蛋白质制备的兔抗血清可以很好地和所表达的蛋白质带特异性结合,且郑引1号小麦幼苗进行干旱处理,提取粗蛋白,SDS-PAGE,蛋白质印迹检测显示,在分子质量28ku处出现特异的蛋白质条带,这说明所制备的抗血清可以与小麦叶片所表达的dehydrin蛋白特异性结合,证明其具有良好的免疫原性.

Expression and Purification of a Dehydrin Gene of Wheat and Preparation of Its Antibody

Dehydrins accumulate during the late stages of embryo genesis or in response to ABA application,low temperature,drought,or any environmentally imposed dehydrative force.Despite the abundance and widespread occurrence in cell of dehydrins,the biochemical role of dehydrins remains elusive.In order to study the expression and functional characteristics of dehydrin gene during different growth,and make the polycolonal antibody,using wheat plumelet under the water deficit condition as experiment material,and its total RNA was extracted.The target dehydrin gene through RT-PCR was got,connect to the cloning vector PUCM-T,recombination expression plasmid PET-32a( ) -wzy1-1 was constructed according to recombination of cloned vector PUCM-T-WZY1-1 of wheat dehydrin gene in this experiment.Then the recombination was transformed into the host strain E.coli BL21(DE3) ,was induced by IPTG and target protein was got.The expression production was detected by SDS-PAGE,and Western blot assays revealed that the fusion protein was expressed in soluble form with a relative molecular mass 37 ku,and the fusion protein was expressed at high level.After purification by Ni-NTA resin affinity chromatography and electroelution in bagfilter,the fusion protein was used to induce the production of polyclonal antibody in rabbits and ELISA detection showed the antigenicity of the fusion protein was satisfactory,Western blot showed the antiserum raised against the recombination dehydrin protein in rabbits could react to the protein expressed specifically,and the protein of zheng yin 1# wheat under drought stress was extracted,through the SDS-PAGE,Western blot assays revealed that there is a protein band with a relative molecular mass 28 ku,this revealed that antiserum could react to the dehydrin protein expressed in wheat leaf specifically and demonstrated its good antigenicity.