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EDS1蛋白是植物寄主SA信号转导通路中具有重要调控功能的蛋白,为获得效价高和选择性强的EDS1抗体,根据NCBI GenBank中报道的EDS1蛋白一级结构信息,采用Blastn、Blastx和ExPASy等生物信息学软件进行序列同源性分析,获得三段序列特异性较高的多肽,并从中优选一段序列特异性多肽,采用9-氟甲氧羰基固相合成法获得序列特异性最好的多肽,采用HPLC和GC-MS测定合成多肽的浓度和分子量,试验表明目的多肽纯度达89.37%,目的多肽分子量为1978.33。采用碳化二亚胺法将多肽与KLH进行偶联获得免疫原——Pep-KLH,并将其免疫新西兰大白兔以获得抗血清和多克隆抗体,采用间接-ELISA和Western blotting测定其效价和特异性,经间接-ELISA检测表明抗血清和多克隆抗体可与Pep发生特异性免疫反应,经Western blotting试验表明抗血清和多克隆抗体可识别烟草叶片特异性条带,其相对分子量为70kD,与预测分子量相符,表明利用该方法制备的EDS1多肽抗体具有较高特异性和灵敏度。
EDS1 protein is an important regulatory function in plant host SA signal transduction pathway. To obtain high titer and selectivity EDS1 antibody, according to the primary structure information of EDS1 protein reported in NCBI GenBank, Blastn, Blastx and ExPASy And other bioinformatics software for sequence homology analysis, to obtain three high sequence-specific peptides, and from the sequence of a specific sequence-specific peptides, using 9-fluoromethoxycarbonyl solid phase synthesis was the best sequence-specific The concentration and molecular weight of the synthesized polypeptide were determined by HPLC and GC-MS. The purity of the polypeptide was 89.37% and the molecular weight of the target polypeptide was 1978.33. Polypeptide was conjugated with KLH by carbodiimide method to obtain immunogen-Pep-KLH, which was immunized with New Zealand white rabbits to obtain antiserum and polyclonal antibody. The titer of the antibody was determined by indirect-ELISA and Western blotting. Specificity, indirect-ELISA test showed that the antiserum and polyclonal antibodies specific immune response with Pep, Western blotting experiments showed that antiserum and polyclonal antibodies can identify tobacco leaf-specific bands, the relative molecular weight of 70kD, Consistent with the predicted molecular weight, it indicates that the EDS1 polypeptide antibody prepared by the method has higher specificity and sensitivity.