香蕉果实转录因子MaWRKY1基因的原核表达和多克隆抗体制备

Prokaryotic Expression of Banana Transcription Factor MaWRKY1 Gene and Preparation of Its Polyclonal Antibody

MaWRKY1是从香蕉果实中克隆出的一个转录因子基因。为进一步研究该基因的功能,制备了MaWRKY1多克隆抗体。选取MaWRKY1基因全长中N端第168~400个氨基酸之间的包括两个WRKYGQK保守域的cDNA序列,构建了原核表达载体pET-MaWRKY1,并转化到大肠杆菌BL21中诱导表达菌体蛋白。SDS-PAGE电泳检测结果表明,His-MaWRKY1融合蛋白成功获得了高效表达,分子量在26kD左右。His-MaWRKY1融合蛋白经过Ni-NTA琼脂糖凝胶树脂纯化,SDS-PAGE制备胶割胶富集,电洗脱法纯化后得到的纯化蛋白浓度达到0.5mg·mL-1。经对新西兰兔进行5次免疫,获得了多克隆抗血清,采用免疫吸附方法对抗血清进行了纯化。将纯化后的抗体通过间接酶联免疫（ELISA）和蛋白质印迹（Western blot）分析,表明所制备的抗体具有很好的效价,效价比为1︰160000,同时具有良好的灵敏度和特异性。进一步提取香蕉不同组织总蛋白,Westernblot检测显示,在分子量26kD左右处出现特异的蛋白质条带,证明所制备的抗体可以与香蕉WRKY蛋白特异性结合,并且低温可以诱导香蕉果实中MaWRKY1蛋白表达,暗示MaWRKY1蛋白表达可能与果实耐冷性有一定的关系。

To investigate the function of the MaWRKY1 gene,a transcription factor previously cloned from banana fruit,the polyclonal antibody of MaWRKY1 was prepared. The recombinant plasmid pET-MaWRKY1 was constructed with 699 bp segments started from the 168 amino acid to the 400 amino acid including the two conserved motif WRKYGQK and an about 26 kD expressed His-MaWRKY1 fusion protein was identified by SDS-PAGE following the expression of the recombinant His-MaWRKY1 fusion protein in E.coli BL21（DE3）cells with high efficiency. About 0.5 mg · mL-1 fusion protein was obtained after puified by Ni-NTA agarosecolumn,separated by SDS-PAGE,excised from the gel and electroeluted, then the purified fusion protein was used as antigen to prepare polyclonal antiserum in rabbits. After the fifthinjection of antigen,the antiserum was obtained and further purified with method of immuno-precipitation. Indirect ELISA test and the western blot analysis showed that the obtained anti-MaWRKY1 polyclonal antibody have good sensitivity and high specificity with a titer of 1︰160 000. Furthermore,western blot assay revealed that a 26 kD peptide was specifically detected in the total proteins of different banana tissues with anti-MaWRKY1 polyclonal antibody,indicating that the antibody could react to the total proteins expressed in banana specifically. In addition,low temperature could induce the expression of MaWRKY1 protein in banana fruit,suggesting that the expression of MaWRKY1 protein in banana fruit was likely to be involved in regulating the chilling-tolerance of fruit.