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根据NCBI GenBank中报道的NPR1一级结构信息,采用Blastn、Blastx、ExPASy和Protean等软件进行序列同源性和抗原性指数分析,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LC-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88%、目的多肽分子量为1.92234 kD。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ELISA和Western blotting测定其效价和特异性,经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65 kD,与预测分子量相符,表明利用该方法制备的NPR1多肽抗体具有较高特异性和灵敏度。
According to the NPR1 primary structure information reported in NCBI GenBank, sequence homology and antigenicity index analysis were carried out by Blastn, Blastx, ExPASy and Protean software to obtain three sequence-specific polypeptides, and a sequence-specific The polypeptide with the best sequence specificity was obtained by solid phase synthesis of 9-fluoromethoxycarbonyl. The concentration and molecular weight of the synthesized polypeptide were determined by HPLC and LC-MS. The purity of the polypeptide was 88%, and the molecular weight of the target polypeptide was 1.92234 kD. Peptide was conjugated with KLH by carbodiimide method to obtain the immunogen Pep-KLH, which was immunized with New Zealand white rabbits to obtain antiserum and polyclonal antibodies. The titer and specificity were determined by ELISA and Western blotting. ELISA showed that antiserum and polyclonal antibodies could specifically immunoreact with Pep. Western blotting showed that antisense and polyclonal antibodies could recognize tobacco leaf-specific bands with a relative molecular weight of 65 kD, which was consistent with the predicted molecular weight. It indicates that the NPR1 polypeptide antibody prepared by this method has high specificity and sensitivity.